Columbia  (MnitJers^ttp  \^\{o 
int^fCitpoflfttigork 

College  of  ^tP^iciansi  anb  ^Uvgtbnsi 


l^ibrarp 


Presented  by 

.DR.  WILLIy\A1  J.  OIES  j;' 

ft  to  enrich  the  library  resourci: 


ava//a/)/e  to  holders 
"^ll  ofthe 

GlES  FELLOWSHIP 

1/2  Biolosicdil  Chemistry 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 
Columbia  University  Libraries 


http://www.archive.org/details/practicalphysiol1916hawk 


PRACTICAL 
PHYSIOLOGICAL  CHEMISTRY 

HAWK 


Absorption  Spectra, 


Oxyhaemoglobin. 


Haemoglobin. 


Carboxy- 
haemoglobln. 


Neutral  Met- 
haemogtobln. 


Alkaline  Met- 
haemoglobln. 


Alkali 
Haematin. 


Absorption  Spectra. 


■^  % 


Reduced  Alkali 
Haematin  or 
Haemochromogen. 


Acid  Haematin  in 
ethereal  solution. 


Acid  Haemato- 
porphyrln. 


Alkaline 

Haematopor- 

phyrln. 


Urobilin  or  Hydro- 
bilirubin  In  acid 
solution. 


Urobilin  or  Hydro- 
bilirubin  4n  alkaline 
solution  after  the 
addition  of  zinc 
chloride  solution. 


Bllicyanin  or 
Cholecyanin  in 
alkaline  solution. 


PRACTICAL 


PHYSIOLOGICAL    CHEMISTRY 


A  BOOK  DESIGNED  FOR  USE  IN  COURSES  IN  PRACTICAL 

PHYSIOLOGICAL  CHEMISTRY  IN  SCHOOLS 

OF  MEDICINE  AND  OF  SCIENCE 


BY 
PHILIP  B.  HAWK,  M.  S.,  Ph.  D. 

PROFESSOR  OF  PHYSIOLOGICAL  CHEMISTRY   AND  TOXICOLOGY  IN  THE 
JEFFERSON   MEDICAL   COLLEGE   OF    PHILADELPHIA 


FIFTH  EDITION,  REVISED  AND  ENLARGED 


WITH  TWO  FULL-PAGE  PLATES  OF  ABSORPTION  SPECTRA  IN  COLORS 

FOUR  ADDITIONAL  FULL-PAGE  COLOR  PLATES  AND  ONE 

HUNDRED  AND  SEVENTY-TWO  FIGURES  OF  WHICH 

TWELVE  ARE  IN  COLORS 


PHILADELPHIA 

P.    BLAKISTON'S   SON   &    CO. 

1012  WALNUT  STREET 
1916 


First  Edition,  Copyright,  1907,  by  P.  Blakiston's  Son  &  Co. 
Second  Edition,  Copyright,  1909,  by  P.  Blakiston's  Son  &  Co. 
Third  Edition,  Copyright,  1910,  by  P.  Blakiston's  Son  &  Co. 
FoxniTH  Edition,  Copyright,  191 2,  by  P.  Blakiston's  Son  &  Co. 

Reprinted,  October,  1913  and  May,  1915 
Fifth  Edition,  Copyright,  1916,  by  P.  Blakiston's  Son  &  Co. 


THE. MAPLE. PRESS. YORK. PA 


THESE    PAGES   ARE 

AFFECTIONATELY  DEDICATED 

TO 

MY  PARENTS 

1866-1916 


PREFACE  TO  FIFTH  EDITION 


The  book  has  been  thoroughly  revised  and  in  part  rewritten.  Five 
additional  chapters  have  been  inserted,  in  order  to  increase  the  useful- 
ness of  the  volume  and  to  keep  thoroughly  abreast  with  recent  develop- 
ments in  Physiological  Chemistry.  The  new  Chapters  are,  Chapter  VI 
on  Nucleic  Acids.'and  Nucleoproteins,  Chapter  VIII  on  Gastric  Analysis, 
Chapter  XI  on  Intestinal  Digestion,  Chapter  XVI  on  Blood  Analysis 
and  Chapter  XXVII  on  Metabolism. 

The  Chapter  on  Metabolism  consists  in  large  part  of  directions 
for  typical  metabolism  tests  which  may  be  conducted  by  the  student  in 
order  to  demonstrate  important  principles  of  metabolism. 

The  Chapter  on  Blood  Analysis  includes  the  most  recent  methods 
which  have  played  such  an  important  part  in  adding  to  our  knowledge 
regarding  the  composition  of  the  blood  under  normal  and  pathological 
conditions. 

The  Chapter  on  Gastric  Analysis  includes  a  general  discussion  of 
titratable  acidity  and  hydrogen-ion  concentration  as  well  as  an  expo- 
sition of  the  Fractional  Method  of  Gastric  Analysis  which  was  elabo- 
rated in  the  author's  laboratory  by  Dr.  Martin  E.  Rehfuss,  and  which 
has  been  very  widely  adopted. 

The  Chapter  on  Nucleic  Acids  and  Nucleoproteins  is  a  brief  dis- 
cussion of  basic  facts  accompanied  by  experiments  suitable  for  student 
use. 

In  order  to  facihtate  the  selection  of  tests  and  methods  for  courses 
in  which  but  httle  time  is  devoted  to  the  subject,  the  actual  laboratory 
procedure  involved  in  such  tests  and  methods  as  the  author  considers 
most  important  has  been  printed  in  black  face  type  to  differentiate 
such  tests  and  methods  from  those  which  he  considers  of  less  impor- 
tance, which  are  set  in  smaU  type.  In  certain  instances,  however,  tests 
and  methods  which  the  author  beHeves  to  be  of  first  importance,  but 
which  are  not  so  readily  adapted  to  student  use,  have  been  printed  in 
small  type. 

The  latest  and  best  methods  of  quantitative  analysis  have  been 
introduced  throughout  the  volume.  The  nephelometer  has  been  dis- 
cussed and  certain  nephelometric  methods  described. 

Certain  tests  and  methods  which  had  obviously  outlived  tlieir 
usefulness  have  been  omitted  from  this  edition.     Others  which  the 


Vlll  PREFACE    TO    FIFTH    EDITION 

author  believes  might  well  have  been  omitted  have  been  retained  in 
small  type.  The  author  believes  that  it  is  preferable  that  each  indi- 
vidual instructor  shall  use  his  own  judgment  as  to  which  tests  and 
methods  he  will  omit  from  his  course.  For  this  reason,  in  connection 
with  certain  quantitative  procedures  more  than  a  single  approved 
method  has  been  given. 

Thirty-five  new  illustrations  have  been  incorporated. 

The  author  takes  pleasure  in  acknowledging  the  important  part 
played  by  Dr.  Olaf  Bergeim  in  the  preparation  of  this  new  edition. 
Dr.  Bergeim's  suggestions  and  assistance  have  been  invaluable  in 
connection  with  all  phases  of  the  re\dsion,  as  well  as  in  the  preparation 
of  new  material  for  introduction.  To  Dr.  Martin  E.  Rehfuss  the 
author  is  indebted  for  the  drawings  of  the  microscopical  constituents 
of  the  feces  and  gastric  contents  and  the  curves  which  are  introduced 
in  the  Chapter  on  Gastric  Analysis,  as  well  as  for  timely  suggestions 
as.  to  certain  clinical  aspects  of  fecal  and  gastric  analysis.  Other 
members  of  the  author's  staff  who  have  aided  in  certain  phases  of  the 
revision  are  Drs.  H.  Rodell  Fishback,-Mehdn  A.  Saylor  and  Clarence  A. 
Smith. 

To  Professors  Lafayette  B.  Mendel,  Paul  E.  Howe,  Marshall  P. 
Cram,  A.  P.  Sy  and  J.  C.  Blake,  the  author  is  also  indebted  for  helpful 
suggestions,  and  to  Professors  WiUiam  J.  Gies  and  Walter  Jones  for 
permission  to  insert  selected  experiments  from  their  laboratory  direc- 
tions. The  author  is  under  further  obligation  to  Professor  Jones  for 
crystals  of  hypoxanthine  chloride  and  guanine  chloride  from  which 
were  prepared  the  microphotographs  appearing  in  the  Chapter  on 
Nucleic  Acids  and  Nucleoproteins.  He  also  wishes  to  thank  Professor 
Chester  C.  Fowler  for  permission  to  insert  unpublished  material. 

The  author  is  very  appreciative  of  the  unfailing  courtesy  and  con- 
sideration of  the  publishing  house  of  P.  Blakiston's  Son  &  Co.;  the 
timely  suggestions  of  Mr.  C.  V.  Brownlow,  Mr.  I.  A.  Hagy  and  Mr. 
Horace  G.  White  have  been  very  helpful. 


CONTENTS 

CHAPTER  I 

t  Page 

Enzymes  and  Their  Action i 

CHAPTER  II 
Carbohydrates 19 

CHAPTER  III 
Salivary  Digestion 54 

CHAPTER  IV 
Proteins:  Their  Decomposition  and  Synthesis 63 

CHAPTER  V 
Proteins:  Their  Classification  and  Properties 92 

CHAPTER  VI 
Nucleic  Acids  and  Nucleoproteins 123 

CHAPTER  VII 

Gastric  Digestion 138 

CHAPTER  VIII 
Gastric  Analysis 148 

CHAPTER  IX 
Fats 176 

CHAPTER  X 
Pancreatic  Digestion 185 

CHAPTER  XI 
Intestinal  Digestion 195 

CHAPTER  XII 

Bile 202 

CHAPTER  XIII 
Putrefaction  Products 212 

CHAPTER  XIV 

Feces 221 

ix 


X  CONTENTS 

CHAPTER  XV 

Paqb 

Blood  and  Lymph 245 

CHAPTER  XVI 
Blood  Analysis 270 

CHAPTER  XVII 

Milk 313 

CHAPTER  XVIII 
Epithelial  and  Connectwe  Tissues 330 

CHAPTER  XIX 
Muscular  Tissue .  339 

CHAPTER  XX 
Nervous  Tissue 353 

CHAPTER  XXI 

Urine:  General  Characteristics  of  Normal  and  Pathological 
Urine 359 

CHAPTER  XXII 
Urine:  Physiological  Constituents 369 

CHAPTER  XXIII 
Urine:  Pathological  Constituents 412 

CHAPTER  XXIV 
Urine:  Organized  and  Unorganized  Sediments 457 

CHAPTER  XXV 
Urine:  Calculi 475 

CHAPTER  XXVI 
Urine:  Quantitative  Analysis 479 

CHAPTER  XXVII 

Metabolism 564 

Reagents  and  Solutions 594 

Index 612 


LIST  OF  ILLUSTRATIONS 


Plate 

I.  Absorption  Spectra  1  . 

II.  Absorption  Spectra  J ^^ 

III.  Osazones Opposite  page  22 

IV.  Normal  Erythrocytes  and  Leucocytes Opposite  page  249 

V.  Uric  Acid  Crystals Opposite  page  380 

VI.  Ammonium  Urate Opposite  page  462 

Figure  Page 

1.  Apparatus  for  Quantitative  Determination  of  Catalase 17 

2.  Dialyzing  Apparatus  for  Students'  Use 24 

3.  Einhorn  Saccharometer 31 

4.  Illustrating  Different  Stages  in  Fermentation 31 

5.  One  Form  of  Laurent  Polariscope 32 

6.  Diagrammatic  Representation  of  the  Course  of  the  Light  through 

the  Laurent  Polariscope 33 

7.  Polariscope  (Schmidt  and  Hansch  Model) 34 

8.  Iodoform 42 

9.  Potato  Starch 44 

10.  Bean  Starch 44 

11.  Arrowroot  Starch 44 

12.  Rye  Starch 44 

13.  Barley  Starch 44 

14.  Oat  Starch 44 

15.  Buckwheat  Starch 44 

16.  Maize  Starch 44 

17.  Rice  Starch 44 

18.  Pea  Starch 44 

19.  Wheat  Starch 44 

20.  Microscopical  Constituents  of  Sahva 58 

21.  Glycocoll  Ester  Hydrochloride 72 

22.  Serine 73 

23.  Phenylalanine 74 

24.  Fischer  Apparatus 75 

25.  Tyrosine 76 

26.  Cystine 76 

27.  Histidine  Dichloride 78 

28.  Leucine 80 

29.  Lysine  Picrate 81 

30.  Aspartic  Acid Si 

31.  Glutamic  Acid S^ 

32.  Levo-a-ProUne 84 

33.  Copper  Salt  of  Proline 84 

xi 


XU  LIST    OF   ILLUSTRATIONS 

FiGUBE                                                                       <  PaQB 

34.  Van  Slyke  Amino  Nitrogen  Apparatus 88 

35.  Section  of  Van  Slyke  Apparatus 88 

36.  Coagulation  Temperature  Apparatus 106 

37.  Edestin 109 

38.  Excelsin,  the  Protein  of  the  Brazil  Nut 110 

39.  Guanine  Chloride 135 

40.  Hypoxanthine  Chloride 136 

41.  Normal  and  Pathological  Curves  after  an  Ewald  Meal 148 

42.  Rehfuss  Stomach  Tube 149 

43.  Influence  of  Acid  Introduced  into  the  Normal  Human  Stomach    .  150 

44.  Hydrogen  Ion  Concentration  Chart 157 

45.  Acidity  Curves  of  Normal  Human  Stomach 163 

46.  Acidity  Curves  from  a  Case  of  Hyperacidity 164 

47.  Acidity  and  Protein  Curves  in  Gastric  Carcinoma 164 

48.  Total  Acidity  and  Protein  Curves  in  Benign  Achylia 165 

49.  Microscopical  Constituents  of  the  Gastric  Contents 172 

50.  Beef  Fat 176 

51.  Mutton  Fat 179 

52.  Pork  Fat 181 

53.  Palmitic  Acid 182 

54.  Melting-point  Apparatus 183 

55.  Bile  Salts 205 

56.  Bilirubin  (Hematoidin) 205 

57.  Cholesterol 210 

58.  Taurine 211 

59.  Glycocoll 211 

60.  Ammonium  Chloride 216 

61.  Hematoidin  Crystals  from  Acholic  Stools 222 

62.  Charcot-Leyden  Crystals 225 

63.  Boas'  Sieve 229 

64-69.  Microscopical  Constituents  of  Feces 230-31 

70.  Oxyhemoglobin  Crystals  from  Blood  of  the  Guinea-pig 251 

71.  Oxyhemoglobin  Crystals  from  Blood  of  the  Rat 251 

72.  Oxyhemoglobin  Crystals  from  Blood  of  the  Horse 252 

73.  Oxyhemoglobin  Crystals  from  Blood  of  the  Squirrel 252 

74.  Oxyhemoglobin  Crystals  from  Blood  of  the  Dog 253 

75.  Oxyhemoglobin  Crystals  from  Blood  of  the  Cat 253 

76.  Oxyhemoglobin  Crystals  from  Blood  of  the  Necturus 254 

77.  Effect  of  Water  on  Erythrocytes 261 

78.  Hemin  Crystals  from  Human  Blood 265 

79.  Hemin  Crystals  from  Sheep  Blood 265 

80.  Sodium  Chloride 267 

81.  Bang  Reduction  Flask 281 

82.  Apparatus  for  Epstein's  Sugar  Method 283 

83.  Bloor's  Nephelometer 291 


LIST    OF   ILLUSTRATIONS  XIU 

FiGCRE  Page 

84.  Nephelometer  in  Position,  Showing  Relation  to  Source  of  Light  .  292 

85.  Lenzmann-Kober  Nephelometer 293 

86.  Direct-vision  Spectroscope 296 

87.  Angular-vision  Spectroscope  Arranged  for  Absorption  Analysis      .  297 

88.  Diagram  of  Angular- vision  Spectroscope 297 

89.  Fleischl's  Hemometer 300 

90.  Pipette  of  Fleischl's  Hemometer 301 

91.  Colored  Glass  Wedge  of  Fleischl's  Hemometer 301 

92.  Dare's  Hemoglobinometer 302 

93.  Horizontal  Section  of  Dare's  Hemoglobinometer 303 

94.  Method  of  FilUng  the  Capillary  Observation  Cell  of  Dare's  Hemo- 

globinometer   303 

95.  Thoma-Zeiss  Counting  Chamber 304 

96.  Thoma-Zeiss  Capillary  Pipettes 305 

97.  Ordinary  Ruling  of  Thoma-Zeiss  Counting  Chamber 306 

98.  Zappert's  Modified  Ruling  of  Thoma-Zeiss  Counting  Chamber  .    .  307 

99.  Biirker's  Pipettes,  Mixing  Flasks,  and  Counting  Chamber.    .    .    .  309 

100.  Ruling  of  Burker  Counting  Chamber 310 

loi.  Schema 311 

102.  Burker  Counting  Chamber 312 

103.  Normal  Milk  and  Colostrum 315 

104.  Lactose 318 

105.  Calcium  Phosphate 322 

106.  Centrifuge  Tube  used  in  Babcock  Fat  Method      324 

107.  Croll's  Fat  Apparatus 325 

108.  Soxhlet  Apparatus 326 

109.  Feser's  Lactoscope 326 

no.  Creatine 342 

111.  Xanthine 344 

112.  Hypoxanthine  Silver  Nitrate 351 

113.  Xanthine  Silver  Nitrate 351 

114.  Deposit  in  Ammoniacal  Fermentation 362 

115.  Deposit  in  Acid  Fermentation 363 

116.  Urinometer  and  Cylinder 364 

117.  Beckmann-Heidenhain  Freezing-point  Apparatus  365 

118.  Urea 372 

119.  Urea  Nitrate 374 

120.  Melting-point  Tubes  Fastened  to  Bulb  of  Thermometer  375 

121.  Urea  Oxalate 376 

122.  Pure  Uric  Acid 380 

123.  Creatinine 382 

124.  Creatinine-Zinc  Chloride 385 

125.  Hippuric  Acid 389 

126.  AUantoin  from  Cat's  Urine 392 

127.  Benzoic  Acid 396 


Xiv  LIST    OF    ILLUSTRATIONS 

FiGUBE  Page 

128.  Calcium  Sulphate 405 

129.  "Triple  Phosphate" 408 

130.  Albumoscope 424 

131.  Marsh  Apparatus 449 

132.  The  Purdy  Electric  Centrifuge 457 

133.  Sediment  Tube  for  the  Purdy  Electric  Centrifuge 457 

134.  Calcium  Oxalate 459 

135.  Calcium  Carbonate 459 

136.  Various  Forms  of  Uric  Acid 461 

137.  Acid  Sodium  Urate 462 

138.  Cystine 462 

139.  Crystals  of  Impure  Leucine 463 

140.  EpitheHum  from  Different  Areas  of  the  Urinary  Tract 466 

141.  Pus  Corpuscles 467 

142.  Hyaline  Casts 468 

143.  Granular  Casts 469 

144.  Granular  Casts 47° 

145.  Epithelial  Casts 47° 

146.  Blood,  Pus,  HyaUne  and  EpitheUal  Casts 470 

147.  Fatty  Casts 471 

148.  Fatty  and  Waxy  Casts 471 

149.  Cylindroids 472 

150.  Crenated  Erythrocytes 473 

151.  Human  Spermatozoa 474 

152.  Folin  Fume  Absorber 483 

153.  Duboscq  Colorimeter 486 

154.  155.  Forms  of  Apparatus  used  in  Methods  of  Folin  and  Associates 

for  Determination  of  Total  Nitrogen,  Urea  and  Ammonia   .    .    .   487 

156.  Bock  and  Benedict  Apparatus 489 

157.  Gulick  Micro-oxidation  Flask. 490 

158.  Van  Slyke  and  Cullen  Apparatus 492 

159.  Folin's  Urea  Apparatus 496 

160.  Doremus-Hinds  Ureometer 497 

161.  Marshall's  Urea  Apparatus 498 

162.  Hiifner's  Urea  Apparatus 498 

163.  Folin's  Ammonia  Apparatus 499 

164.  Folin  Improved  Absorption  Tube 500 

165.  Ruhemann's  Uricometer 513 

166.  Hall's  Purinometer 517 

167.  Esbach's  Albuminometer 532 

168.  Scott- Wilson  Apparatus 536 

169.  Blood  Sugar  as  Influenced  by  Diet 566 

170.  Influence  of  Protein  Ingestion  on  Endogenous  Uric  Acid  Output  573 

171.  The  Endogenous  Uric  Acid  Output  during  Fasting 573 

172.  Berthelot-Atwater  Bomb  Calorimeter 586 


PHYSIOLOGICAL  CHEMISTRY 


CHAPTER  I 

ENZYMES  AND  THEIR  ACTION 

According  to  the  old  classiti cation  ferments  were  divided  into  two 
classes,  the  organized  ferments  and  the  unorganized  ferments.  As  organ- 
ized ferments  or  true  ferments  there  were  grouped  such  substances  as 
yeast  and  certain  bacteria  which  were  supposed  to  act  by  virtue  of  vital 
processes,  whereas  the  unorganized  ferments  included  salivary  amylase 
(ptyalin),  gastric  protease  (pepsin),  pancreatic  protease  (trypsin),  etc., 
which  were  described  as  ''non-Hving  unorganized  substances  of  a 
chemical  nature."  Kiihne  designated  this  latter  class  of  substances  as 
enzymes  i^v  ^vfirj — in  yeast).  This  division  into  organized  ferments 
(^true  ferments)  and  unorganized  ferments  (enzymes)  was  generally 
accepted  and  was  practically  unquestioned  until  Buchner  overthrew 
it  in  the  year  1897  by  his  epoch-making  investigations  on  zymase. 
Previous  to  this  time  many  writers  had  expressed  the  opinion  that  the 
action  of  the  ferment  organisms  was  similar  to  that  of  the  unorganized 
ferments  or  enzymes  and  that  therefore  the  activity  of  the  former  was 
possibly  due  to  the  production  of  a  substance  in  the  cell,  which  was  in 
nature  similar  to  an  enzyme.  Investigation  after  investigation,  how- 
ever, failed  to  isolate  any  such  principle  from  an  active  cell  and  the 
exponents  of  the  "vital"  theory  became  strengthened  in  their  belief  that 
certain  fermentative  processes  brought  about  by  living  cells  could  not 
occur  apart  from  the  biological  activity  of  such  cells.  However,  as 
early  as  1858,  Traube  had  enunciated,  in  substance,  the  principles 
which  were  destined  to  be  fundamental  in  our  modern  theory  of  fermen- 
tation. He  expressed  the  belief  that  the  yeast  cell  produced  a  product 
in  its  metabolic  activities  which  had  the  property  of  reacting  with  sugar 
with  the  production  of  carbon  dio.xide  and  alcohol,  and  further  that  this 
reaction  between  the  product  of  the  metabolism  of  the  yeast  cell  and  the 
sugar  occurred  without  aid  from  the  original  cell.  It  was  not  until  1897, 
however,  that  this  theory  was  placed  upon  a  firm  experimental  basis. 
This  was  brought  about  through  the  efforts  of  Buchner,  who  succeeded  in 
isolating  from  the  living  yeast  cells  a  substance  (zymase)  which,  when 
freed  from  the  last  trace  of  organized  cellular  material,  was  able  to  bring 


2  PHYSIOLOGICAL   CHEMISTRY 

about  the  identical  fermentative  processes  which,  up  to  this  time,  had 
been  deemed  possible  only  in  the  presence  of  the  active,  living  yeast  cell. 

Buchner's  manipulation  of  the  yeast  cells  consisted  in  first  grind- 
ing them  with  sand  and  infusorial  earth,  after  which  the  finely  divided 
material  was  subjected  to  great  pressure  (300  atmospheres)  and  yielded 
a  liquid  which  possessed  the  fermentative  activity  of  the  unchanged 
yeast  cell.^  This  Hquid  contained  zymase,  the  principal  enzyme  of 
the  yeast  cell.  Later  the  lactic-acid-  and  acetic-acid-producing  bac- 
teria were  subjected  by  Buchner  to  treatment  similar  to  that  accorded 
the  yeast  cells,  and  the  active  intracellular  enzymes  were  obtained. 
Many  other  instances  are  on  record  in  which  a  soluble,  active  agent  has 
been  isolated  from  ferment  cells,  with  the  result  that  it  is  pretty  well 
estabhshed  that  all  the  so-called  organized  ferments  elaborate  sub- 
stances of  this  character. 

Enzymes  act  by  catalysis  and  hence  may  be  termed  catalyzers  or 
catalysts.  A  simple  rough  definition  of  a  catalyst  is  "a  substance 
which  alters  the  velpcity  of  a  chemical  reaction  without  undergoing 
any  apparent  physical  or  chemical  change  itself  and  without  becoming 
a  part  of  the  product  formed."  It  is  a  well-known  fact  that  the  veloc- 
ity of  the  greater  number  of  chemical  reactions  may  be  changed 
through  the  presence  of  some  catalyst.  For  example,  take  the  case  of 
hydrogen  peroxide.  It  spontaneously  decomposes  slowly  into  water 
and  oxygen.  In  the  presence  of  colloidal  platinum, ^  however,  the  de- 
composition is  much  accelerated  and  ceases  only  when  the  destruction 
of  the  hydrogen  peroxide  is  complete.  Without  multiplying  instances, 
sufl&ce  it  to  say  that  there  is  a  close  analogy  between  inorganic  catalysts 
and  enzymes,  the  main  point  of  difference  between  the  enzymes  and  most 
of  the  inorganic  catalysts  being  that  the  enzymes  are  colloids.  The 
great  majority  of  enzymes  are  hydrolytic  in  character. 

We  may  define  an  enzyme  as  an  organic  catalyst  which  is  elaborated 
by  an  animal  or  vegetable  cell  and  whose  activity  is  entirely  independent 
of  any  of  the  life  processes  of  such  a  cell.  According  to  this  definition 
the  enzyme  zymase  elaborated  by  the  yeast  cell  is  entirely  comparable 
to  the  enzyme  pepsin  elaborated  by  the  cells  of  the  stomach  mucosa. 
One  is  derived  from  a  vegetable  cell,  the  other  from  an  animal  cell,  yet 
the  activity  of  neither  is  dependent  upon  the  integrity  of  the  cell. 

Inasmuch  as  each  of  the  enzymes  has  an  action  which  is  more  or  less 
specific  in  character,  and  since  it  is  a  fairly  simple  matter,  ordinarily,  to 
determine  the  character  of  that  action,  the  classification  of  the  enzymes 

*  In  later  investigations  the  process  was  improved  by  freezing  the  ground  cells  with 
liquid  air  and  finely  pulverizing  them  before  applying  the  pressure. 

2  Produced  by  the  passage  of  electric  sparks  between  two  platinum  terminals  immersed 
in  distilled  water,  thus  liberating  ultra-microscopic  particles. 


ENZYMES   AND    THEIR    ACTION  3 

is  not  attended  with  very  great  difficulties.  They  are  ordinarily  classi- 
fied according  to  the  nature  of  the  substrate^  or  according  to  the  type 
of  reaction  they  bring  about.  Thus  we  have  various  classes  of  enz}-mes, 
such  as  amylolytic,'^  proteolytic,  lipolytic,  glycolytic,  uricolytic,  autolytic, 
oxidizing,  reducing,  inverting,  protein-coagulating,  deamidizing,  etc.  In 
every  instance  the  class  name  indicates  the  individual  type  of  enzy- 
matic activity  which  the  enzymes  included  in  that  class  are  capable  of 
accomplishing.  For  example,  amylolytic  enzymes  facilitate  the  hydro- 
lysis of  starch  (amylum)  and  related  substances,  lipolytic  enzymes 
facihtate  the  hydrolysis  of  fats  (Xittos),  whereas  through  the  agency  of 
uricolytic  enzymes  uric  acid  is  broken  down.  There  is  a  tendency, 
at  the  present  time,  to  harmonize  the  nomenclature  of  the  enzymes  by 
the  use  of  the  termination  -ase.  According  to  this  system  of  nomen- 
clature, all  starch-transforming  enzymes,  or  so-called  amylolytic  en- 
zymes, are  called  amylases;  all  fat-splitting  enz3'mes  are  called  lipases, 
etc.  Thus  ptyalin,  the  amylolytic  enzyme  of  the  saliva,  would  be 
termed  salivary  amylase  in  order  to  distinguish  it  from  pancreatic  amy- 
lase (amylopsin)  and  vegetable  amylases  (diastase,  etc.).  According 
to  the  same  system,  the  fat-splitting  enzyme  of  the  gastric  juice  would 
be  termed  gastric  lipase  to  differentiate  it  fro-^n  t>ancreatic  lipase  (steap- 
sin),  the  fat-splitting  enzyme  of  the  pancreatic  juice. 

Defensive  (protective)  enzymes  are  those  believed  to  be  manufac- 
tured by  certain  cells  (perhaps  the  leucocytes)  and  passed  into  the 
circulating  blood  in  order  to  digest  any  foreign  material  of  endogenous 
or  exogenous  origin  that  may  have  found  its  way  into  the  circulation. 
The  most  important  defensive  enzymes  are  proteolytic  enzymes. 
Abderhalden^  claims  that  the  parenteral  introduction  of  any  foreign 
protein  into  the  animal  body  will  be  followed  by  the  appearance  in  the 
blood  of  a  defensive  enzyme  capable  of  digesting  that  protein.  He  also 
claims  that  in  pregnancy  the  passage  into  the  blood  of  protein  material 
in  the  form  of  cells  and  fragments  of  chorionic  villi  will  cause  the 
appearance  in  the  blood  of  a  defensive  proteolytic  enzyme  capable  of 
digesting  placenta  protein.  The  AbderJialden  reaction  for  pregnancy 
is  based  upon  this  hypothesis.  The  reaction  has  been  widely  employed 
and  much  has  been  said  both  for  and  against  its  accuracy.^  Modifications 
of  the  reaction  have  been  employed  as  an  aid  in  the  diagnosis  of  various 
disorders,  e.g.,  cancer,   tuberculosis,  dementia   pra^cox,  etc.     Taylor^ 

^  Substance  acted  upon.     See  Lippmann:  Ber.  d.  Deutsch.  Chcm.  Ges.,  36,  331,  1903. 

2  Armstrong  suggests  the  use  of  the  termination  "clastic"  instead  of  "lytic."  He  calls 
attention  to  the  fact  that  amylolytic,  in  analogy  with  electrolytic,  means  "decomposition  by 
means  of  starch"  and  is  therefore  a  misnomer.  He  suggests  the  use  of  aniyloclastic, 
proleoclaslic,  etc. 

^  Abwelirfennentc  dcs  tierischcn  Organismus,  5th  Ed.,  Berlin,  1915,  Springer. 

^Bronfenbrenner:  Jour.  Am.  Med.  Assn.,  65,  1268,  1915.  Van  Slyke:  Neiu  York  Med. 
Jour.,  103,  219,  1916. 

^  Taylor:  Jour.  Biol.  Chcm.,  22,  59,  191 5. 


4  PHYSIOLOGICAL   CHEMISTRY 

has  very  recently  shown  that  "the  rabbit  does  not  form  a  protective 
ferment  in  response  to  the  injection  of  the  protamine  of  the  salmon. " 
He,  however,  points  out  the  fact  that  this  does  not  prove  that  a  de- 
fensive enz}Tne  is  not  formed  in  response  to  the  passage  of  placental 
protein  into  the  blood. 

Our  knowledge  regarding  the  distribution  of  enzymes  has  been 
wonderfully  broadened  in  recent  years.  Up  to  within  a  few  years, 
the  real  scientihc  information  as  to  the  enzymes  of  the  animal  organism, 
for  example,  was  limited,  in  the  main,  to  a  rather  crude  understanding 
of  the  enzymes  intimately  connected  with  the  main  digestive  func- 
tions of  the  organism.  We  now  have  occasion  to  beheve  that  enzymes 
are  doubtless  present  in  every  animal  cell  and  are  actively  associated 
with. all  \-ital  phenomena.  As  a  preeminent  example  of  such  cellular 
acti\-ity  may  be  cited  the  liver  cell  with  its  reputed  complement  of  15-20 
or  more  enzymes. 

A  list  of  the  more  important  enz}-mes  together  with  their  class,  dis- 
tribution, substrate  and  end-products  is  given  below. 

CLASSIFICATION  OF  ENZYMES 


Name  and  Class  Distribution  Substrate  End-products 


Carbohydrases "-^  i  Carbohydrates j 

I.  Amylases Starch,  dextrin,  etc 

(a)  Pancreatic. . . .  Pancreatic  juice Starch,  dextrin,  etc Maltose. 

(amylopsin') 

(b)  Salivary Saliva Starch,  dextrin,  etc Maltose. 

(ptyalin) 

(c)  Vegetable Malt,  rice  fungus,  etc Starch,  dextrin,  etc Maltose. 


2.  Glycogenase. . . . 

Liver,  muscles  ? . . . . 

Glycogen 

.  .  .  .  Dextrin      and      maltose 
(glucose?) 

3.  Inulase 

Fungi,  other  plants. 

Inulin 

.  .  .  .  Fructose. 

4.  Lactase 

Intestinal  juice  and 

mucosa 

Lactose 

.  .  .  .  Glucose  and  galactose. 

5.   Maltase 

Blood  serum,  Uver,  saliva, 
pancreatic    and    intestinal 
juices  and  lymph. 

Maltose 

.  .  .  .  Glucose. 

6.  Sucrase 

(invertasei 

Intestinal  juice  and 

mucosa. 

Sucrose 

.  .  .  .  Glucose  and  fructose. 

7.  Zymase 

Yeast 

Sugars 

.  .  .  .  Alcohol,  CQj,  etc. 

Carboxylase 

Yeast 

COOH    group 
acids. 

of  aUphatic  Carbon  dioxide. 

Amino  compounds ... 

I.  Adenase 

Adenine 

.  .  .  .  Hypoxanthine. 

2.  Arginase 

Intestine,      liver, 
spleen,  etc. 

kidney, 

Arginine 

.  .  .  .  Ornithine  and  urea. 

3.   Guanase 

Animal  tissues 

Guanin 

.  .  .  .  Xanthine. 

4.   Urease 

Micrococcus       ureae,       soy 
bean,  etc. 

Urea 

.  .  .  .  Carbon  dioxide  and  am- 
monia. 

Glucosidases 

1.  Emulsin 

2.  Invertase 

Plants 

Yeast,  etc 

Glucosides  (amygdalin 
others). 

/3-glucosides 

o-glucosides 

and 

.  .  .  .  Glucose,  etc. 
. . . .  Glucose,  etc. 

ENZYMES    AND    THEIR   ACTION  ; 

CLASSIFICATION  OF  E'SZYMES.— Continued 
Name  and  Class  Distribution  Substrate  End-products 

Lipases Fats 

1.  Autolytic Animal  tissues Fats.  P'atty  acid  an^i  n.v,_^.-rv.i. 

2.  Pancreatic Pancreatic  juice Fats.  Fatty  acid  and  glycerol. 

(steapsin)  . ,        ,    , 

3.  Vegetable Castor  bean,  etc Fats Fatty  acid  and  glycerol. 


Nucleases N'ucleicacidand  derivatives. 

1.  Nucleicacidase. .  Intestinal  mucosa  and  juice.  Nucleic  acid N'ucleotides. 

other  tissues. 

2.  Nucleotidase.. .  .  Intestinal  mucosa  and  juice.  Nucleotides Phosphoric  acid  and  nu- 

other  tissues.  cleosides. 

3.  Nucleosidase.. .  .  Tissues Nucleosides Carbohydrate  and  bases. 

Oxidases. 

1.  Laccase Lac  tree,  fungi,  etc Polyhydric  para-phenols  as  Oxidation  products. 

hydroquinol     and      pyro- 
gallol. 

2.  Peroxidase Plant  and  animal  tissues. .  .  Organic  pero.xides Oxygen  or  oxidation  prod- 

ucts. 

3.  Calalase Plant  and  animal  tissues..  .  Hydrogen  peroxide Oxygen  or  oxidation  prod- 

ucts. 

4.  Purine-oxidases.  Purines. 

(a)  Hypoxanthine  Animal  tissues Hypoxanthine.  Xanthine. 

oxidase. 

(b)  Uricase Animal  tissues Uric  acid .V.lantoin. 

(c)  Xanthine  Animal  tissues Xanthine Uric  acid. 

oxidase. 

5.  Tyrosinase Plant  and  animal  tissues..  .  Tyrosine.  Homogentisic  acid,  etc. 

Peptases Polypeptids 

I .  Erepsin Intestinal  mucosa  and  juice.  Peptids Simpler       peptids       and 

other  tissues.  amino  acids. 

Phytase Rice  bran,  liver,  blood Phytin Inositol   and    phosphoric 

acid. 


Proteases.  Proteins 

1.  Coagulases .' Proteins  in  solution 

(a^   Rennin Gastric  juice Casein Paracasein. 

(gastric) 

(b)  Rennin Pancreatic  juice Casein Paracasein. 

(pancreatic) 

(c)  Thrombin Blood ■  Fibrinogen. .  .    .  Fibrin. 

2.  Pepsin Gastric  juice Proteins Proteoses,  peptones,  and 

(acid-protease)  peptides. 

3.  Trypsin. ./.....  Pancreatic  juice Proteins Proteoses,  peptones,  pep- 

(alkali-protease) ;  tides,  amino-acids. 

4.  Vegetable      pro-j 
teases. 

(a)  Bromelin Pineapple Proteins..  Proteoses,  peptones,  etc. 

(b)  Papain. Pawpaw Proteins..  Proteoses,  peptones,  etc. 

(papayotin) 

Purinases(see  Purine 
Oxidases  and  Pur- 
ine Deaminases). 


In  text-book  discussions  of  the  enzymes  it  is  customary  to  say  that 
very  little  is  known  regarding  the  chemical  characteristics  of  these  sub- 
stances since  no  member  of  the  enzyme  group  has,  up  to  the  present 
time,  been  prepared  in  an  absolutely  pure  condition.  Apparently,  how- 
ever, from  the  nature  of  the  facts  in  the  case,  it  would  be  much  more 
accurate  to  say  that  we  absolutely  do  not  know  whether  a  specific  enzyme 
has,  or  has  not,  been  prepared  in  a  pure  state.  (Some  authors,  like 
Arthus,  have  assumed  that  enzymes  are  not  chemical  individuals,  but 
properties  conferred  upon  bodies.)  The  enzymes  are  very  difticult  to 
prepare  in  anything  like  a  condition  approximating  purity,  since  they 
are  very  prone  to  change  their  nature  during  the  process  by  which  the 


O  PHYSIOLOGICAL    CHEMISTRY 

investigator  is  attempting  to  isolate  them.  For  this  reason  we  have 
absolutely  no  proof  that  the  final  product  obtained  is,  or  is  not,  in  the 
same  state  of  purity  it  possessed  in  the  original  cell.  Some  of  the  en- 
zymes are  more  or  less  closely  associated  with  the  proteins  from  the  fact 
that  they  are  both  formed  in  every  cell  as  the  result  of  the  cellular  ac- 
tivity, both  may  be  removed  from  solution  by  *' salting-out,"  both  are 
for  the  most  part  non-diffusible  and  are  probably  very  similar  as  re- 
gards elementary  composition.  Hence  in  the  preparation  of  some 
enzymes  it  is  extremely  difficult  to  make  an  absolute  separation  from 
the  protein.  Most  of  the  evidence  points  to  the  protein  character  of 
enzymes.^  Under  certain  conditions  enzymes  are  readily  adsorbed  by 
shredded  protein  material,  such  as  fibrin,  and  may  successfully  resist 
the  most  prolonged  attempts  at  washing  them  free.  We  may  sum- 
marize some  of  the  properties  of  the  great  body  of  enzymes  as  follows : 
Enzymes  are  soluble  in  dilute  glycerol,  sodium  chloride  solution, 
dilute  alcohol  and  water,  and  precipitable  by  ammonium  sulphate 
and  strong  alcohol.  Their  presence  may  be  proven  froin  the  nature 
of  the  end-products  of  their  action  and  not  through  the  agency  of  any 
chemical  test.  They  are  colloidal  and  non-diffusible,  and  occur  closely 
associated  with  protein  material  with  which  they  generally  possess  many 
properties  in  common.  Each  enzyme  shows  the  greatest  activity  at  a 
certain  temperature  called  the  optimum  temperature;  there  is  also  a 
minimum  and  a  maximum  temperature  for  each  specific  enzyme.  Their 
action  is  inhibited  by  sufficiently  lowering  the  temperature,  although 
some  activity  may  be  shown  at  o°  or  even  at  lower  temperatures 
and  freezing  does  not,  in  most  cases,  permanently  injure  enzymes. 
Most  enzymes,  if  in  solution,  are  entirely  destroyed  by  subjecting 
them  to  a  temperature  of  70-1  oo°C.  The  best  known  enzymes,  whether 
derived  from  warm-blooded  or  cold-blooded  animals,  are  most  active 
between  35°-45°C.  The  nature  of  the  surrounding  media  alters  the 
velocity  of  the  enzymatic  action,  some  enzymes  being  more  active  in 
acid  solution  whereas  others  require  an  alkahne  fluid. 

Many  of  the  more  important  enzymes  do  not  occur  preformed 
within  the  cell,  but  are  present  in  the  form  of  a  zymogen  or  mother- 
substance.  In  order  to  yield  the  active  enzyme  this  zymogen  must  be 
transformed  in  a  certain  specific  manner  and  by  a  certain  specific  sub- 
stance. This  transformation  of  the  inactive  zymogen  into  the  active 
enzyme  is  termed  activation.  For  instance,  the  zymogen  of  the  enzyme 
pepsin  of  the  gastric  juice,  termed  pepsinogen,  is  activated  by  the  hydro- 
chloric acid  secreted  by  the  gastric  cells  (see  page  141),  whereas  the  actir 
vation  of  the  trypsinogen  of  the  pancreatic  juice  is  brought  about  by  a 

^  Others  seem  to  be  like  the  substrate  on  which  they  act,  e.g.,  carbohydrate. 


ENZYMES   AND    THEIR    ACTION  7 

substance  termed  enterokinase^  (see  page  196).  These  are  examples  of 
many  well-known  activation  processes  going  on  continually  within  the 
animal  organism.  The  agency  which  is  instrumental  in  activating  a 
zymogen  is  generally  termed  a  zymo-exciter  or  a  kinase.  In  the  cases 
cited  hydrochloric  acid  would  be  termed  a  zymo-exciter  and  entero- 
kinase  would  be  termed  a  kinase. 

After  filtering  yeast  juice,  prepared  by  the  Buchner  process  (see  page 
2),  through  a  Martin  gelatin  filter,  Harden  and  Young  showed  that 
the  colloids  left  behind  and  the  filtrate  were  both  inactive  fermenta- 
tively.  Upon  treating  the  colloid  material  (enz>Tne)  with  some  of  the 
filtrate,  however,  the  mixture  was  shown  to  be  able  to  bring  about  pro- 
nounced fermentation.  It  is  believed  that  a  co-enzyme  present  in  the 
filtrate  was  the  efficient  agent  in  the  transformation  of  the  inactive 
enzyme.  It  is  necessary  to  make  frequent  renewals  of  the  co-enzyme 
in  order  to  maintain  continuous  fermentation.  It  was  further  shown 
that  this  co-enzyme,  in  addition  to  being  diffusible,  was  not  destroyed 
by  boiling  and  that  it  disappeared  from  yeast  juice  when  this  latter 
was  fermented  or  allowed  to  undergo  autolysis.  The  exact  nature  of 
this  co-enzyme  of  zymase  is  unknown.  The  co-enzyme  action,  in  this 
case,  is  probably  dependent  upon  the  presence  of  two  individual 
agencies,  one  of  which  is  phosphates. 

It  has  been  shown  by  Loevenhart  that  the  property  of  acting  as  a 
pancreatic  lipase  co-enzyme  is  vested  in  hile  salts,  and  ]VIagnus  has 
further  shown  that  the  synthetic  salts  are  as  efficient  in  this  regard  as 
the  natural  ones.  A  few  other  instances  of  co-enzyme  demonstrations 
have  been  reported. 

Electrolytes  are  very  important  factors  in  facilitating  or  inhibiting 
enzyme  action.^  For  example,  the  CI  ion  in  proper  amount  facihtates 
the  action  of  amylases.^  In  fact  the  presence  of  the  CI  or  Br  ion  is 
apparently  absolutely  essential  to  the  activity  of  pancreatic  amylase, 
inasmuch  as  dialysis  renders  this  enzyme  inactive,  the  activity  return- 
ing on  the  addition  of  sodium  chloride.^  The  acidity  or  hydrogen  ion 
concentration  of  the  solution  also  exerts  much  influence  on  the  activity 
of  enzymes.  It  has  been  demonstrated  in  the  case  of  certain  enz>Tnes, 
at  least,  that  the  continuous  vibration  or  shaking  of  their  solutions  tends 
to  produce  a  destruction  of  the  enzyme.  Ultraviolet  light  also  has  a 
destructive  action  on  enzymes. 

The  so-called  "specificity"  of  enzyme  action  is  an  interesting  and 
important  fact.     That  enzymes  are  very  specific  as  to  the  character  of 

1  According  to  Delezenne,  trypsinogen  may  be  rapidly  activated  by  soluble  calcium  salts. 
-  For  literature,  see  Kendall  and  Sherman:  Jour.  Am.  Client.  Soc,  32,  1087,  1910. 
'  Wohlgemuth:  Biochemisclte  Zeilschrifl,  q,  10,  1908. 
*  Bierry:  Ibid.,  40,  357,  191 2. 


8  PHYSIOLOGICAL    CHEMISTRY 

the  substrate,  or  substance  acted  upon,  is  well  known.  Emil  Fischer 
investigated  this  problem  of  specificity  extensively  in  connection  with 
the  fermentation  of  sugars  and  reached  the  conclusion  that  enzymes, 
with  the  possible  exception  of  certain  oxidases,  can  act  only  upon  such 
substances  as  have  a  specific  stereo-isomeric  relationship  to  themselves. 
He  considers  that  the  enzyme  and  its  substrate  must  have  an  inter- 
relation, such  as  the  key  has  to  the  lock,  or  the  reaction  does  not  occur. 
Fischer  was  able  to  predict,  in  certain  definite  cases,  from  a  knowledge 
of  the  constitution  and  stereo-chemical  relationships  of  a  substance, 
whether  or  not  it  would  be  acted  upon  by  a  certain  enzyme.  An  appli- 
cation of  this  specificity  of  enzyme  action  may  be  seen  in  the  well-known 
facts  that  certain  enzymes  act  on  carbohydrates,  others  on  fats,  and 
others  on  protein;  and,  moreover,  that  the  group  of  those  which  trans- 
form carbohydrates,  for  example,  is  further  subdivided  into  specific  en- 
zymes each  of  which  has  the  power  of  acting  alone  upon  some  one  sugar. 

It  has  been  conclusively  shown,  in  the  case  of  certain  enzymes,^ 
at  least,  that  their  action  is  a  reversible  one  and  is,  in  all  its  main  fea- 
tures, directly  analogous  to  the  reversible  reactions  produced  by  chem- 
ical means.  For  instance,  in  the  saponification  of  ethyl-butyrate  by 
means  of  pancreatic  lipase,  it  has  been  shown  that  upon  the  formation 
of  the  end-products  of  the  reaction,  i.e.,  butyric  acid  and  ethyl  alcohol, 
there  is  reversion^  and  the  reaction  is  stationary.  This  does  not  mean 
there  are  no  chemical  changes  going  on,  but  simply  indicates  that 
chemical  equilibrium  has  been  established,  and  that  the  change  in  one 
direction  is  counterbalanced  by  the  change  in  the  opposite  direction. 
Pancreatic  lipase  was  one  of  the  first  enzymes  to  have  the  reversibility 
of  its  reaction  clearly  demonstrated.^  A  knowledge  of  the  fact  that 
lipase  possesses  this  reversibility  of  action  is  of  extreme  physiological 
importance  and  aids  us  materially  in  the  explanation  of  the  processes 
involved  in  the  digestion,  absorption,  and  deposition  of  fats  in  the 
animal  organism  (see  page  178). 

Euler^  claims  that  enzymatic  cleavage  and  synthesis  are  often  brought 
about  by  two  different  components  of  an  enzyme  preparation.  He 
would  indicate  this  fact  by  giving  the  termination  -ese  to  those  enzymes 
exerting  a  synthetic  function.  For  example,  the  enzyme  which  catalyzes 
the  formation  of  nitriles  Euler  would  call  nitrile^e  in  distinction  from 
nitrila^e  which  splits  nitriles.     He  would  further  designate  as  phos- 

'  This  is  probably  a  general  condition. 

^  The  re-synthesis  of  ethyl-butyrate  from  its  hydrolysis  products.     This  may  be  indi- 
cated thus: 

C3H7COO.C2H5+H20fc>C3H7COOH+C2H50H. 

Elhyl-biUyrale.        Butyric  acid.      Ethyl  alcohol. 
3  This  principle  was  first  demonstrated  in  connection  with  the  enzyme  maltase  (seep.  57). 
*  Euler:  Zcilschrijljiir  physiologische  chemie,  74,  13,  191 1. 


ENZYMES    AND    THEIR    ACTION  9 

phatese  the  enzyme  which  builds  up  phosphoric  acid  esters  of  carbo- 
hydrates in  distinction  from  phosphatase  which  causes  their  cleavage. 
In  the  same  way  he  would  differentiate  the  lipolytic  enzymes  into  lipases 
and  lipeses. 

In  respect  to  many  enzymes  it  has  been  found  that  the  law  govern- 
ing the  action  of  inorganic  catalyzers  is  directly  applicable,  i.e.,  that 
the  intensity  is  almost  directly  proportional  to  the  concentration  of  the 
enzyme.  In  the  case  of  enzymes,  however,  there  is  a  difference  in  that  a 
maximum  intensity  is  soon  reached  and  that  subsequent  concentration 
of  the  enzyme  is  productive  of  no  further  increase  in  intensity.  The  en- 
zymes which  have  been  shown  to  obey  this  linear  law  are  lipase,  sucrase, 
rennin,  and  tr\'psin.  In  certain  instances,  where  this  law  of  direct 
proportionality  betweien  the  intensity  of  action  and  the  concentration 
of  enzymes  does  not  hold,  it  has  been  found  that  the  Schutz-Borissow 
law,  first  experimentally  demonstrated  by  E.  Schiitz,  was  applicable. 
This  is  to  the  effect  that  the  intensity  is  directly  proportional  to  the 
square  root  of  the  concentration,  or  conversely,  that  the  relative  con- 
centrations of  enzyme  preparations  are  directly  proportional  to  the  squares 
of  the  intensities} 

It  has  been  shown  that  there  are  certain  substances  which  possess 
the  property  of  directly  inhibiting  or  preventing  the  action  of  a  cata- 
lyzer. These  are  called  anti-catalyzers  or  paralyzers  and  have  been  com- 
pared to  the  anti-toxins.  Related  to  this  class  of  anti-catalytic  agents 
stand  the  anti-enzymes.  The  first  anti-enzyme  to  be  reported  was  the 
antirennin  of  Morgenroth.  This  was  produced  by  injecting  into  an 
animal  increasing  doses  of  rennet  solution,  whereupon  an  "anti" 
substance  was  subsequently  found  both  in  the  serum  and  in  the  milk, 
which  prevented  the  enzyme  rennin  from  exerting  its  normal  activity 
in  the  presence  of  casein.  In  other  words,  anti-rennin  had  been 
formed  in  the  serum  of  the  animal,-  through  the  repeated  injections  of 
rennet  solution.  Since  the  discovery  of  this  anti-enz)Tne,  anti-bodies 
have  been  demonstrated  for  pepsin,  trypsin,  lipase,  urease,  amylase, 
laccase,  tyrosinase,  emulsin,  papain,  and  thrombin.  According  to 
Weinland,  the  reason  why  the  stomach  does  not  digest  itself  is.  that 
during  life  there  is  present  in  the  mucous  membrane  of  the  stomach  an 
anti-enzyme  (anti-pepsin)  which  has  the  property  of  inhibiting  the  action 
of  pepsin.  A  similar  substance  (anti-trypsin)  is  present  in  the  intestinal 
mucosa  as  well  as  in  the  tissues  of  various  intestinal  worms.  Some  in- 
vestigators are  not  inclined  to  accept  the  enzyme  nature  of  these 
inhibitory  agents  as  proven. 

^  This  Schutz-Borissow  law  is  not  generally  applicable. 
*  Serum  is  normally  anti-lryplic. 


lO  PHYSIOLOGICAL   CHEMISTRY 

The  investigations  of  Ehrlich^  and  of  Neuberg^  have  served  to  cause 
a  complete  revision  of  our  ideas  regarding  yeast  fermentation.  EhrKch, 
for  example,  has  shown  that  yeast  will  liberate  ammonia  from  amino  acids 
and  leave  behind  a  non-nitrogenous  complex.  Among  these  complexes 
amyl  alcohol,  succinic  acid  and  others  may  be  mentioned.  Thus,  amyl 
alcohol  results  from  the  fermentation  of  leucine,  whereas  ethyl  alcohol 
results  from  the  fermentation  of  sugar.  Neuberg  has  demonstrated  the 
presence  in  the  yeast  of  an  enzyme  termed  carboxylase  which  has  the 
property  of  splitting  of  carbon  dioxide  from  the  carboxyl  group  of  amino 
and  other  aliphatic  acids.  The  findings  mentioned  above  constitute  the 
basis  for  much  important  work  on  so-called  "sugar-free  fermentation." 

For  a  more  extended  consideration  of  enzymes  the  student  is  referred 
to  the  following  sources: 

Bayliss. — The  Nature  of  Enzyme  Action,  Third  Edition,  Long- 
mans, Green  and  Co.,  New  York  and  London. 

CoHNHEiM. — Enzymes,  Wiley  and  Sons,  New  York,  191 2. 

DucLAUX. — Traite  de  Microbiologic,  Masson  and  Co.,  Paris. 

Effront. — Enzymes  and  their  Applications,  Translated  by  Pres- 
cott,  Wiley  and  Sons,  New  York. 

EuLER. — (a)  Allgemeine  Chemie  der  Enzyme,  Bergmann,  Wies- 
baden, 1910.  (b)  Ergebnisse  der  Physiologic,  1909-10.  (c)  General 
Chemistry  of  the  Enzym.es,  Translated  by  Pope,  Wiley  and  Sons, 
1912. 

Oppenheimer. — Die  Fermente  und  Ihre  Wirkungen,  Vierte  Auflage, 
Vogel,  Leipzig. 

Samuely. — Handbuch  der  Biochemie  des  Menschen  und  der  Thiere 
(Oppenheimer),  Gustav  Fischer,  Jena,  1910-11. 

Wohlgemuth. — Grundriss  der  Fermentmethoden,  1913,  Berlin, 
Springer. 

EXPERIMENTS  ON  ENZYMES  AND  ANTI-ENZYMES 
A.  Experiments  on  Enzymes^ 

I.  AMYLASES 

I.  Demonstration  of  Salivary  Amylase.* — ^To  25  c.c.  of  a  i  per  cent  starch 
paste  in  a  small  beaker,  add  5  drops  of  saliva  and  stir  thoroughly.  At  intervals 
of  a  minute  remove  a  drop  of  the  solution  to  one  of  the  depressions  of  a  test-tablet 

'  Ehrlich:  Biochemische  Zeitschrifl,  36,  477,  191 1. 

*  Neuberg  and  Collaborators:  Biochemische  Zeitschrifl,  31, 170;  32,  323;  36  (60,  68,  and 
76).  1911. 

2  If  it  is  deemed  advisable  by  the  instructor  to  give  all  the  practical  work  upon  enzymes 
at  this  point  in  the  course,  additional  experiments  will  be  found  in  Chapters  III,  VI,  VII, 
X  and  XI. 

*  For  a  discussion  of  this  enzyme  see  p.  55. 


ENZYMES    AND   THEIR    ACTION  II 

and  test  by  the  iodine  test.^    If  the  blue  color  with  iodine  still  forms  after  five 
minutes,  add  another  5  drops  of  saliva. 

The  opalescence  of  the  starch  solution  should  soon  disappear, 
indicating  the  formation  of  sohible  starch  {amidtilin)  which  gives  a  blue 
color  with  iodine.  This  body  should  soon  be  transformed  into  erythro- 
dextrin  which  gives  a  red  color  with  iodine,  and  this,  in  turn,  should 
pass  into  achroodextrin  which  gives  no  color  with  iodine.  This  point  is 
called  the  achromic  point.  When  this  point  is  reached  test  by  Fehling's 
test^  to  show  the  production  of  a  reducing  substance  (maltose).  A 
positive  Fehling's  test  may  be  obtained  while  the  solution  still  reacts  red 
with  iodine,  inasmuch  as  some  sugar  is  formed  from  the  soluble  starch 
coincidently  with  the  formation  of  the  erythrodextrin.  For  further 
discussion  of  the  transformation  of  starch  see  page  56. 

2.  Demonstration  of  Pancreatic  Amylase.^ — Proceed  exactly  as  indicated  above 
in  the  Demonstration  of  Salivary  Amylase  except  that  the  saliva  is  replaced  by  5  c.c. 
of  pancreatic  extract  prepared  as  described  on  p.  189.'*  Pancreatic  amylase  trans- 
forms the  starch  in  a  manner  entirely  analogous  to  the  transformation  resulting 
from  the  action  of  salivary  amylase. 

3.  Preparation  of  Vegetable  Amylase. — Extract  finely  ground  malt  with  water, 
filter  and  subject  the  filtrate  to  alcohoUc  fermentation  by  means  of  yeast.  When 
fermentation  is  complete  filter  off  the  yeast  and  precipitate  the  amylase  from  the 
filtrate  by  the  addition  of  alcohol.  The  precipitate  may  be  filtered  off  and  ob- 
tained in  the  form  of  a  fine  white  powder. 

A  purer  preparation^  is  obtained  if  the  solution  is  dialyzed  against 
water  at  about  io°C.  (in  the  ice-box)  for  24  hours,  filtered  and  pre- 
cipitated with  alcohol  or  acetone.  First  alcohol  or  acetone  to  make  a  50 
per  cent  solution  is  added,  the  precipitate  thus  formed  being  rejected, 
while  the  precipitate  formed  on  the  addition  of  sufficient  alcohol  or 
acetone  to  make  a  final  concentration  of  65-70  per  cent  is  preserved,  and 
dried  in  a  vacuum  desiccator  at  a  low  temperature. 

4.  Demonstration  of  Vegetable  Amylase. — This  enzyme  may  be  demon- 
strated according  to  the  directions  given  under  Demonstration  of  SaUvary  Amylase, 
page  10,  with  the  exception  that  the  sahva  used  in  that  experiment  is  replaced  by 
an  aqueous  solution  of  the  vegetable  amylase  powder  prepared  as  described 
above.  ^ 

^  See  p.  45. 

*  See  p.  26. 

^  For  a  discussion  of  this  enzyme  see  p.  187. 

*  Commercial  preparations  of  pancreatic  amylase  may  be  substituted  for  the  pancreatic 
extract. 

'Sherman  and  Schlesinger:  /.  Am.  Ch.  Soc,  35,  1617,  1915. 

^  If  desired  the  first  aqueous  extract  of  the  original  malt  may  be  used  in  this  demonstra- 
tion.    Commercial  taka-diaslase  may  also  be  employed. 


12  PHYSIOLOGICAL    CHEMISTRY 


n.  PROTEASES 


1.  Preparation  of  Gastric  Protease. ^ — Treat  the  finely  comminuted  mucosa  of 
a  pig's  stomach  with  0.4  per  cent  hydrochloric  acid  and  extract  at  38°C.  for 
24-48  hours.  The  filtrate  from  this  mixture  constitutes  a  very  satisfactory  acid 
extract  of  gastric  protease  (see  page  144). 

2.  Demonstration  of  Gastric  Protease. — Introduce  some  protein  material 
(fibrin,  coagulated  egg-white,  or  washed  lean  beef)  into  the  acid  extract  of  gastric 
protease  prepared  as  above  described,'-  add  an  equal  volimie  of  0.4  per  cent 
hydrochloric  acid  and  place  the  mixture  at  38°C.  for  2-3  days.  Identify  the 
products  of  digestion  according  to  directions  given  on  page  144. 

Carmine- fibrin  may  also  be  used  in  this  test.  This  is  prepared 
by  running  fibrin  through  a  meat  chopper  washing  carefully  and  placing 
in  a  }4  per  cent  ammoniacal  carmine  solution  (very  little  excess  am- 
monia should  be  present)  until  the  maximum  coloration  of  the  fibrin 
(a  dark  red)  is  obtained.  The  fibrin  is  then  washed  in  water  and  water 
acidified  with  acetic  acid.     It  is  preserved  under  glycerol. 

To  15  c.c.  of  the  solution  to  be  tested  add  a  small  amount  of  the 
carmine  fibrin  and  allow  to  digest  at  room  temperature.  Digestion  will 
be  shown  by  the  setting  free  of  carmine  with  coloration  of  the  solution. 
This  is  a  delicate  test  for  pepsin.  A  control  should  be  run  using  acid  of 
same  strength  as  that  of  enzyme  solution  tested. 

3.  Preparation  of  Pancreatic  Protease.^ — A  satisfactory  extract  of  this 
enzyme  may  be  made  from  the  pancreas  of  a  pig  or  sheep  according  to  the  direc- 
tions given  on  page  189. 

4.  Demonstration  of  Pancreatic  Protease. — Into  an  aUcaUne  extract  of  pan- 
creatic protease,^  prepared  as  directed  on  page  189,  introduce  some  fibrin,  coagu- 
lated egg-white  or  lean  beef  and  place  the  mixture  at  38°C.  for  2-5  days.*  At 
the  end  of  that  period  separate  and  identify  the  end-products  of  the  action  of  pan- 
creatic protease  according  to  the  directions  given  on  page  189. 

Congo-red  fibrin  may  be  used  in  this  test.  This  may  be  prepared 
by  placing  fibrin  in  faintly  alkaline  congo-red  solution  and  heating  to 
8o°C.  The  fibrin  is  then  washed.  A  small  amount  of  this  colored 
fibrin  is  placed  in  the  slightly  alkaline  solution  of  the  enzyme.  Diges- 
tion is  shown  by  a  red  coloration  of  the  solution  due  to  setting  free  of 
Congo  red. 

'  Also  called  pepsin,  pepsase,  gastric  protease  and  acid  protease.  For  a  discussion  of  this 
enzyme  see  p.  141. 

^  If  so  desired,  a  solution  of  commercial  pepsin  powder  in  0.2  per  cent  hydrochloric  acid 
may  be  substituted. 

^  .\lso  called  trypsin,  irypsase,  pancreatic  protease  and  alkali  protease.  For  a  discussion 
of  this  enzyme  see  p.  186. 

^  A  0.25  per  cent  sodium  carbonate  solution  of  commercial  trypsin  or  pancreatin  may  be 
substituted. 

.  5  A  few  c.c.  of  toluol  or  an  alcoholic  solution  of  thymol  should  be  added  to  prevent  putre- 
faction. 


ENZYMES    AND   THEIR    ACTION  1 3 

5.  Demonstration  of  a  Vegetable  Protease. — A  commercial  preparation  of 
papain  {papayotin,  carase  or  papase),  the  protease  of  the  fruit  of  the  pawpaw  (carica 
papaya),  may  be  used  in  this  connection.  Follow  the  same  procedure  as  that  de- 
scribed under  Gastric  Protease  (see  p.  12). 

It  has  been  demonstrated  by  IMendel  and  Blood'  that  the  presence  of  HCN 
will  accelerate  the  proteolytic  activity  of  papain.  It  is  suggested  that  the  HCN 
acts  as  a  so-called  co-enzyme  (see  page  7). 

Vines-  believes  that  "papain"  consists  of  a  mixture  of  two  enzymes,  a  pepsin 
and  an  erepsin.  Mendel  and  Blood  do  not  consider  the  evidence  on  this  point  as 
conclusive. 

m.  LIPASES 

1.  Preparation  of  Pancreatic  Lipase.* — An  extract  of  this  enzyme  may  be 
prepared  from  the  pancreas  of  the  pig  or  sheep  according  to  the  directions  given 
on  page   189.' 

2.  Demonstration  of  Pancreatic  Lipase. — Into  each  of  two  test-tubes  intro- 
duce 10  c.c.  of  milk  and  a  small  amount  of  litmus  powder.  To  the  contents  of  one 
tube  add  3  c.c.  of  a  neutral  extract  of  pancreatic  lipase  and  to  the  contents  of  the 
other  tube  add  3  c.c.  of  a  boiled  neutral  extract  of  pancreatic  lipase.  Keep  the 
tubes  at  38°C.  and  watch  for  color  changes. 

The  blue  color  of  the  litmus  powder  will  gradually  give  place  to  a 
red.  This  change  in  color  of  the  litmus  from  blue  to  red  has  been 
brought  about  by  the  fatty  acid  which  has  been  produced  through  the 
lipolytic  action  exercised  by  the  lipase  upon  the  milk  fats. 

3.  Preparation  of  Vegetable  Lipase. — This  enzyme  may  be  readily  prepared 
from  castor  beans,  several  months'  old,  by  the  following  procedure:*  Grind  the 
shelled  beans  very  fine^  and  extract  for  twenty-four-hour  periods  with  alcohol-ether 
and  ether,  in  turn.  Reduce  the  semi-fat-free  material  to  the  finest  possible  consist- 
ency by  means  of  mortar  and  pestle  and  pass  this  material  through  a  sieve  of  very 
fine  mesh.  Place  this  material  in  a  Soxhlet  extractor  and  extract  for  one  week. 
This  fat-free  powder  may  then  be  used  to  demonstrate  the  action  of  vegetable 
lipase.  Powder  prepared  as  described  may  be  used  in  quantitative  tests.  For 
ordinary  qualitative  tests  it  is  not  necessary  to  remove  the  last  traces  of  fat  and 
therefore  the  extraction  period  in  the  Soxhlet  apparatus  may  be  much  shortened. 

4.  Demonstration  of  Vegetable  Lipase. — The  lipolytic  action  of  the  lipase  pre- 
pared from  the  castor  bean,  as  just  described,  may  be  demonstrated  in  a  manner 
entirely  analogous  to  that  used  in  the  Demonstration  of  Pancreatic  Lipase,  see 
above.  Proceed  as  indicated  in  that  experiment  and  substitute  the  vegetable 
lipase  powder  for  the  neutral  extract  of  pancreatic  lipase.  The  type  of  action  is 
entirely  analogous  in  the  two  instances. 

^  Mendel  and  Blood:  Journal  of  Biological  Chemistry,  8,  177,  1910. 

''Vines:  Annals  of  Botany,  19,  174,  1905. 

'  Also  called  steapsin.  P'or  a  discussion  of  the  enzyme  see  p.  188.  .V  very  active  lipo- 
lytic extract  may  also  be  prepared  from  the  liver. 

*  If  preferred,  a  glycerol  extract  may  be  prepared  according  to  the  directions  given  by 
Kanitz  {Zeitschrift  fiir  physiologische  Chemie,  1906,  46,  p.  482)  or  commercial  pancrealin 
may  be  employed. 

^  A.  E.  Taylor:  On  Fermentation;   University  of  California  Publications,  igo-;. 

^  The  shells  should  be  removed  without  the  use  of  water.  These  beans  are  poisonous, 
due  to  their  content  of  ricin. 


14  PHYSIOLOGICAL   CHEMISTRY 

An  experiment  similar  to  Experiment  2,  page  193,  may  also  be  tried  if  desired. 
In  this  experiment  0.2  c.c.  of  either  ethyl  buiyrate  or  aniyl  acetate  may  be  employed. 

IV.  INVERTASESi 

1.  Preparation  of  Vegetable  Sucrase.- — Thoroughly  grind  about  100  grains  of 
brewer's  yeast  in  a  mortar  with  sand.  Spread  the  ground  yeast  in  thin  layers  on 
glass  or  porous  plates  and  dry  it  rapidly  in  a  current  of  dry,  warm  air.  Powder 
this  dry  yeast,  extract  it  with  distilled  water  and  filter.  Pour  the  filtrate  into 
acetone,  stir  and  after  permitting  the  acetone  mixture  to  stand  for  a  few  minutes 
filter  on  a  Buchner  funnel.  The  resulting  precipitate,  after  drying  and  pulver- 
izing, may  be  used  to  demonstrate  vegetable  sucrase. 

2.  Demonstration  of  Vegetable  Sucrase. — To  about  5  c.c.  of  a  i  per  cent 
solution  of  sucrose  in  a  test-tube  add  a  small  amount  of  the  sucrase  powder  pre- 
pared as  directed  above.  Place  the  tube  at  38°C.  for  24-72  hours  and  at  the  end 
of  that  period  test  the  solution  by  Fehling's  test.  Reduction  indicates  that  the 
active  sucrase  powder  has  transformed  the  non-reducing  sucrose  into  glucose 
and  fructose,  and  these  sugars,  in  turn,  have  reduced  the  Fehling  solution. 

For  other  experiments  on  Invertases,  see  Chapter  XI. 

V.  OXIDASES  3 

I.  Demonstration  of  Oxidase.- — Oxidases  or  oxidizing  enzymes  con- 
stitute a  very  important  group  of  intracellular  enzymes.  They  are 
intimately  connected  with  the  oxidation  processes  in  the  plant  and  ani- 
mal organisms. 

1.  Cut  a  thin  slice  from  a  freshly  pared  potato,  place  it  on  a  watch  glass  and 
examine  at  intervals  during  the  laboratory  exercise.  Note  that  the  colorless 
potato  gradually  becomes  brown. 

This  is  due  to  the  oxidation  of  para-oxyphenyl  substances  such  as 
tyrosin,  in  the  cells  and  in  the  intracellular  juice  of  the  potato.  Two 
oxidases  which  have  the  power  of  accelerating  the  oxidation  of  para- 
oxyphenyl  compounds  are  called  tyrosinase  and  laccase. 

2.  Preparation  of  Potato  Extract. — Scrape  a  pared  potato  by  means  of  a  knife 
or  scalpel  or  comminute  the  potato  substance  by  means  of  a  grater.  Extract  the 
macerated  potato  substance  by  means  of  water.  Strain  through  cheese  cloth  and 
filter  the  extract.  Make  an  iodine  test  on  the  solid  substance  (see  Starch,  page 
45),  and  save  the  water  extract  for  use  in  the  following  experiments. 

3.  Oxidation  of  Para-oxyphenyl  Compounds  by  Potato  Oxidases. — Introduce 
5  c.c.  of  filtered  potato  extract  prepared  as  indicated  above,  into  each  of  six  test- 
tubes.  Introduce  additional  reagents  into  the  tubes  according  to  the  following 
series : 

(a)  Potato  extract  +  5  drops  of  toluol  (control). 

(b)  Potato  extract  +  5  drops  of  ether  (control). 

*  The  inverting  enzymes  of  the  alimentary  tract;  Mendel  and  Mitchell:  American  Journal 
of  Physiology,  20,  81,  1907-08. 

2 For  a  discussion  of  this  enzyme  see  p.  195. 

2  These  experiments  have  been  adapted  from  directions  contained  in  the  Laboratory 
Notes  of  Professor  Gies  of  the  College  of  Physicians  and  Surgeons,  New  York. 


ENZYMES    AND    THEIR    ACTION  1 5 

(c)  Potato  extract  +  5  drops  of  i  per  cent  phenol  solution. 

(d)  Potato  extract  +  5  drops  of  i  per  cent  '*  tri-cresol  "  solution. 

(e)  Potato  extract  (boiled  and  cooled)  +  5  drops  of  i  per  cent  phenol  solution, 

(f)  Potato  extract  (boiled  and  cooled)  -f-  5  drops  of  i  per  cent  "tri-cresol" 
solution. 

Shake  the  contents  of  the  six  tubes  thoroughly.  Are  there  any  immediate 
color  changes?  Place  the  tubes  in  your  rack,  and  examine  them  at  the  next 
laboratory  exercise. 

4.  Experiments  with  Typical  Oxidase  Reagents. — Introduce  5  c.c.  of  filtered 
potato  extract  into  each  of  four  test-tubes.     Add  oxidase  reagents  as  follows : 

(a)  Potato  extract  +  10  drops  of  guaiac  solution.^ 

(b)  Potato  extract  +  10  drops  of  a-naphthol  solution. - 

(c)  Potato  extract  +10  drops  of  para-phenylenediamine  hydrochloride 
solution.^ 

(d)  Potato  extract  +  5  drops  of  a-naphthol  solution  +  5  drops  of  para- 
phenylenediamine  hydrochloride  solution  +  5  drops  of  10  per  cent  sodium 
carbonate  (Indophenol  Test). 

Shake  the  contents  of  each  tube  thoroughly  and  note  immediate  color 
changes.  Place  the  tubes  in  the  rack  and  leave  them  undisturbed  until  the 
end  of  the  laboratory  exercise.  Note  any  changes  or  pecuUarities  in  the  color- 
ation efifects,  especially  at  the  surface  of  the  liquid. 

In  tube  (a)  the  guaiaconic  acid  of  the  guaiac  resin  has  been  oxidized 
and  formed  guaiac  blue. 

In  tube  (b)  a  violet  coloration  due  to  the  production  of  di-naphthol 
appears.     The  oxidase  has  oxidized  the  a-naphthol. 

In  tube  (c)  we  have  a  change  whose  chemistry  is  not  well  known. 

In  tube  (d)  we  have  the  production  of  indophenol  from  the  a-naph- 
thol and  the  para-phenylenediamine  hydrochloride  under  the  influence  of 
oxidase.  The  indophenol  is  soluble  in  the  alkaline  solution.  The  color 
gradually  changes  from  red  to  purple  as  the  indophenol  accumulates. 

The  production  of  the  above  colors  does  not  possess  any  biological 
significance.  These  colors  simply  serve  to  indicate  that  certain  reac- 
tions are  taking  place  which  occur  normally  in  living  cells,  although 
in  the  latter  case  they  are  of  course  unaccompanied  by  any  color 
change.  Intracellular  oxidase  favors  the  utilization  of  oxygen  by  a 
cell,  just  as  the  potato  oxidase  has  facilitated  the  oxidation  of  the 
chromogens  in  the  above  tests. 

VI.  CATALASE 

Demonstration  of  Catalase. — The  various  animal  tissues  as  liver, 

kidney,  blood,  lung,  muscle  and  brain  contain  an  enzyme  called  catalase 

or  peroxidase  which  possesses  the  property  of  decomposing  hydrogen 

1  Made  by  dissolving  0.5  gram  of  guaiac  resin  in  30  c.c.  of  95  per  cent  alcohol. 
^  Made  by  dissolving  i  gram  of  a-naphthol  in  100  c.c.  of  95  per  cent  alcohol. 
^  Dissolve  I  gram  of  para-phenylene  diamine  hydrochloride  in  100  c.c.  of  water. 


1 6  PHYSIOLOGICAL    CHEMISTRY 

peroxide  as  well  as  certain  biological  peroxides  with  the  liberation  of 
oxygen.  Catalase  is  an  indirect  oxidizing  enzyme,  that  is,  it  brings  about 
oxidations  only  in  the  presence  of  a  peroxide.  Catalase  is  also  found  in 
many  plant  tissues  and  an  extract  of  it  may  be  prepared  from  potatoes. 

1.  Vegetable  Catalase. — Into  each  of  four  test-tubes  place  5  c.c.  of  filtered 
potato  extract  prepared  as  in  Experiment  2,  page  14.  Prepare  a  second 
series  of  four  tubes  (see  4,  p.  15),  but  use  a  boiled  potato  extract.  Prepare 
also  a  third  series  using  water  instead  of  potato  extract.  Now  add  to  each 
of  the  twelve  tubes  5  drops  of  a  3  per  cent  solution  of  hydrogen  peroxide.  While 
the  resultant  Uvely  effervescence  is  in  progress  add  to  each  series  the  four 
"Typical  Oxidase  Reagents"  in  the  order  and  quantities  specified  in  the  preceding 
experiment (4).  Allow  the  tubes  to  remain  undisturbed  and  carefully  note  com- 
parative effects  during  the  remainder  of  the  laboratory  exercise.^ 

2.  Animal  Catalase. — The  presence  of  this  enzyme  may  also  be  demonstrated 
as  follows:  Introduce  into  a  low,  broad,  wide-mouthed  bottle  some  pulped  liver 
tissue  and  a  porcelain  crucible  containing  neutral  hydrogen  peroxide.^  Connect  the 
bottle  with  a  eudiometer  filled  with  water,  upset  the  crucible  of  hydrogen  peroxide 
upon  the  liver  pulp  and  note  the  collection  of  gas  in  the  eudiometer.  This  gas  is 
oxygen  which  has  been  liberated  from  the  hydrogen  peroxide  through  the  action  of 
the  catalase  of  the  liver  tissue. 

See  next  experiment  for  a  method  for  the  quantitative  determination  of  catalase 
based  on  the  above  principle. 

3.  Quantitative  Determination  of  Catalase.^ — In  the  determination  of  the 
catalase  values  of  tissues  weighed  portions  of  the  tissue  under  examination  should 
be  ground  with  sand  in  a  mortar  then  treated  with  four  volumes  of  chloroform  water 
and  permitted  to  extract  for  24  hours  at  room  temperature.  An  apparatus  such 
as  that  shown  in  Fig.  i  may  be  employed  in  determining  the  catalase  values.  The 
main  features  of  the  apparatus  are  based  upon  those  of  a  dehvery  funnel  for  intro- 
ducing liquids  under  increased  or  diminished  pressure. 

In  making  a  determination  introduce  a  measured  volume  (1-4  c.c.)  of  the  filtered 
extract^  into  the  small  flask  and  insert  the  modified  Johnson  burette  graduated  to 
5  c.c.  and  containing  50  c.c.  of  hydrogen  peroxide  (Oakland  dioxygen  neutral^  to 
Congo  red)  into  the  neck  of  the  flask.  Fill  the  eudiometer  with  water  and  place  in 
position.  Close  cocks  A  and  C  and  open  cocks  B  and  D  thus  permitting  5  c.c.  of 
the  peroxide  to  flow  into  the  flask.  Shake  the  contents  of  the  flask  briskly^  and 
record  the  volume  of  oxygen  evolved  in  a  two-minute  period  taking  readings  at 
intervals  of  fifteen  seconds. 

Calculation. — When  a  series  of  comparative  tests  are  made  on  different  tissues 
or  on  the  same  tissue  under  different  conditions  it  is  considered  satisfactory  to  make 
a  comparison  of  the  catalase  values  upon  the  basis  of  the  volume  of  oxygen  evolved 

^  This  experiment  has  been  adapted  from  one  contained  in  the  Laboratory  Notes  of  ' 
Professor  Gies  of  the  College  of  Physicians  and  Surgeons,  New  York. 

^  Mendel  and  Leavenworth:  American  Journal  of  Physiology,  21,  85,  1908. 
^  Hawk:  Journal  of  American  Chemical  Society,  33,  425,  igii. 

*  If  less  than  4  c.c.  of  extract  are  used  the  volume  should  be  made  up  to  4  c.c.  bj'  the 
addition  of  distilled  water. 

^  An  acid  reactioji  modifies  the  rate  of  the  oxygen  evolution.  (See  Mendel  and  Leaven- 
worth, American  Journal  of  Physiology,  21,  85,  1908.) 

*  In  making  a  series  of  comparative  tests  it  is  essential  that  the  shaking  process  should  be 
uniform  from  determination  to  determination. 


ENZYMES    AND    THEIR   ACTION 


17 


in  a  period  of  two  minutes  from  5  c.c.  of  neutral  hydrogen  peroxide  by  means  of  i  c.c. 
of  a  I  :  4.  chloroform-water  extract  of  the  tissue. 

B.  Experiments  on  Anti-Enzymes 

I,  F>reparation  of  an  Extract  of  Anti -Pepsin. ' — Grind  up  a  number  of  intestinal 
worms  (ascaris)-  with  quartz  sand  in  a  rnortar.  Subject  this  mass  to  high  pressure, 
filter  the  resultant  juice  and  treat  it  with  alcohol  until  a  concentration  of  60  per 
cent  is  reached.  If  any  precipitate  forms  it  should  be  filtered  off^  and  alcohol 
added  to  the  filtrate  until  the  concentration  of  alcohol  is  85  per  cent,  or  over.  The 
anti-enzyme  is  precipitated  by  this  concentration.     Permit  this  precipitate  to  stand 


Fig.  I. — .\i>PARATUs  for  QuANTixAxrvE  Determination  of  Catalase. 


for  twenty-four  hours,  then  filter  it  off,  wash  it  with  95  per  cent  alcohol,  absolute 
alcohol,  and  ether,  in  turn,  and  finally  dry  the  substance  over  sulphuric  acid.  The 
sticky  powder  which  results  may  be  used  in  this  form  or  may  be  dissolved  in  water  as 
desired  and  the  aqueous  solution  used.'' 

2.  Demonstration  of  Anti-Pepsin.^ — Introduce  into  a  test-tube  a  few  fibrin 
shreds  and  equal  volumes  of  pepsin-hydrochloric  acid®  and  ascaris  extract  made  as 
indicated  above.  Prepare  a  control  tube  in  which  the  ascaris  extract  is  replaced  by 
water.  Place  the  tubes  at  38°C.  Ordinarily  in  one  hour  the  fibrin  in  the  control 
tube  will  be  completely  digested.  The  fibrin  in  the  tube  containing  the  ascaris 
extract  may,  however,  remain  unchanged  for  days,  thus  indicating  the  inhibitory 
influence  exerted  by  the  anti-enzyme  present  in  this  extract. 

^  Anti-gastric-protease  or  anti-acid-protease. 

*  These  may  be  readily  obtained  from  pigs  at  a  slaughter  house. 

^  This  precipitate  consists  of  impurities,  the  anti-enzyme  not  being  precipitated  until  a 
higher  concentration  of  alcohol  is  reached. 

*  The  original  ascaris  extract  possesses  much  greater  activity  than  either  the  powder  or 
the  aqueous  solution. 

^  ^lartin  H.  Kischer:  Physiology  of  Alitmntalion.  1007,  p.  134. 

*  Made  by  bringing  0.015  gram  of  pepsin  into  solution  in  7  c.c.  of  water  and  0.23  gram 
of  concentrated  hvdrochloric  acid. 


1 8  PHYSIOLOGICAL    CHEMISTRY 

3.  Preparation  of  an  Extract  of  Anti -Trypsin.' — The  extract  may  be  prepared 
from  the  intestinal  worm,  ascaris,  according  to  the  directions  given  on  page  17. 

4.  Demonstration  of  Anti-Trypsin. — Introduce  into  a  test-tube  a  few  shreds 
of  fibrin  and  equal  volumes  of  an  artificial  tryptic  solution^  and  the  ascaris  extract 
made  as  described  on  page  17.  Prepare  a  control  tube  in  which  the  ascaris 
extract  is  replaced  by  water.     Place  the  two  tubes  at  38°C. 

Ordinarily  the  fibrin  in  the  control  tube  will  be  completely  digested 
in  two  hours.  The  fibrin  in  the  tube  containing  the  ascaris  extract  may, 
however,  remain  unchanged  for  days,  thus  indicating  the  inhibitory 
influence  of  the  anti-enzyme. 

Blood  serum  also  contains  anti-try psin.  This  may  be  demonstrated 
as  follows:  Introduce  equal  volumes  of  serum  and  artificial  tryptic 
solution  (prepared  as  described  above)  into  a  test-tube  and  add  a  few 
shreds  of  fibrin.  Prepare  a  control  tube  containing  boiled  serum.  Place 
the  two  tubes  at  38°C.  It  will  be  observed  that  the  fibrin  in  the  tube 
containing  the  boiled  serum  digests,  whereas  that  in  the  other  tube  does 
not  digest.  The  anti-trypsin  present  in  the  unboiled  serum  has  exerted 
an  inhibitory  influence  upon  the  action  of  the  trypsin. 

C.  Quantitative  Applications 

Methods  for  the  quantitative  determination  of  various  enzymes 
will  be  found  in  the  following  chapters:  Amylase  (Chapter  X); 
Erepsin  (Chapter  XI) ;  Pepsin  (Chapter  VIII) ;  Trypsin  (Chapter  X) ; 
Urease  (Chapter  XXVI). 

^  Anti-pancreatic-protease  or  anti-alkali-protease. 

^Made  by  dissolving  0.04  gram  of  sodium  carbonate  and  0.015  pram  of  trypsin  in  8 
c.c.  of  water. 


CHAPTER  II 
CARBOHYDRATES 

The  name  carbohydrates  is  given  to  a  class  of  bodies  which  are  an 
especially  prominent  constituent  of  plants  and  which  are  found  also  in 
the  animal  body  either  free  or  as  an  integral  part  of  various  proteins. 
They  are  called  carbohydrates  because  they  contain  the  elements  C,  H 
and  O;  the  H  and  0  being  present  in  the  proportion  to  form  w^ater.  The 
term  is  not  strictly  appropriate  inasmuch  as  there  are  bodies,  such  as 
acetic  acid,  lactic  acid  and  inositol,  which  have  H  and  O  present  in  the 
proportion  to  form  water,  but  which  are  not  carbohydrates,  and  there 
are  also  true  carbohydrates  which  do  not  have  H  and  O  present  in  this 
proportion,  e.g.,  rhamnose,  C6H12O5. 

Chemically  considered,  the  carbohydrates  are  aldehyde  or  ketone 
derivatives  of  complex  alcohols.  Treated  from  this  standpoint,  the 
aldehyde  derivatives  are  spoken  of  as  aldoses,  and  the  ketone  derivatives 
are  spoken  of  as  ketoses.  The  carbohydrates  are  also  frequently  named 
according  to  the  number  of  oxygen  atoms  present  in  the  molecule,  e.g., 
trioses,  pentoses,  and  hexoses. 

The  more  common  carbohydrates  may  be  classified  as  follows: 

I.  Monosaccharides. 

1.  Pentoses,  C5H10O5. 

{a)  Arabinose. 

(b)  Xylose. 

(c)  Rhamnose  (Methyl-pentose),  CeHioOs- 

2.  Hexoses,  CeH^Oe. 

(a)  Glucose. 

(b)  Fructose. 

(c)  Galactose. 

II.  Disaccharides,  CisHooOu- 

1.  Maltose. 

2.  Lactose. 

3.  Iso-Maltose. 

4.  Sucrose. 

,       III.  Trisaccharides,  C18H32O16. 
I.  Raffinose. 

19 


20  PHYSIOLOGICAL   CHEMISTRY 

IV.  Polysaccharides,  (C6Hio05)x- 

1.  Gum  and  Vegetable  Mucilage  Group. 

(a)  Dextrin. 

(b)  Vegetable  Gums. 

2.  Starch  Group. 

(a)  Starch. 
{b)   Inulin. 

(c)  Glycogen. 

(d)  Lichenin. 

3.  Cellulose  Group. 

{a)  Cellulose. 

(b)  Hemicelluloses. 

(i)  Pentosans. 

Gum  Arabic. 
(2)  Hexosans. 

Galactans. 
Agar-agar. 

Each  member  of  the  above  carbohydrate  classes,  except  the  members 
of  the  pentose  group,  may  be  supposed  to  contain  the  group  CeHioOs, 
called  the  saccharide  group.  The  polysaccharides  consist  of  this  group 
alone  taken  a  large  number  of  times,  whereas  the  disaccharides  may  be 
supposed  to  contain  two  such  groups  plus  a  molecule  of  water,  and  the 
monosaccharides  to  contain  one  such  group  plus  a  molecule  of  water. 
Thus,  (CeHioOs).^  =  polysaccharide,  (CeHioOsji  +  HoO  — ^  disacchar- 
ide,  CeHioOs  +  H2O  -^  monosaccharide.  In  a  general  way  the  solu- 
bility of  the  carbohydrates  varies  with  the  number  of  saccharide  groups 
present,  the  substances  containing  the  largest  number  of  these  groups 
being  the  least  soluble.  This  means  simply  that,  as  a  class,  the  mono- 
saccharides (hexoses)  are  the  most  soluble  and  the  polysaccharides 
(starches  and  cellulose)  are  the  least  soluble. 

MONOSACCHARIDES 

Hexoses,  CeHiiOe 

The  hexoses  are  monosaccharides  containing  six  oxygen  atoms  in  a 
molecule.  They  are  the  most  important  of  the  simple  sugars,  and  two 
of  the  principal  hexoses,  glucose  and  fructose,  occur  widely  distributed 
in  plants  and  fruits.  Of  these  two  hexoses,  glucose  results  from  the 
hydrolysis  of  starch,  whereas  both  glucose  and  fructose  are  formed  in 
the  hydrolysis  of  sucrose.  Galactose,  which  with  glucose  results  from 
the  hydrolysis  of  lactose,  is  also  an  important  hexose.  These  three 
hexoses  are  fermentable  by  yeast,  and  yield  levulinic  acid  upon  heating 


CARBOHYDRATES  2 1 

with  dilute  mineral  acids.  They  reduce  metallic  oxides  in  alkaline 
solution,  are  optically  active  and  extremely  soluble.  With  phenyl- 
hydrazine  they  form  characteristic  osazones. 

CH2OH 

GLUCOSE   (CH0H)4 

j 
CHO 

Glucose,  also  called  dextrose  or  grape  sugar,  is  present  in  the  blood 
in  small  amount  and  also  occurs  in  traces  in  normal  urine. ^  After 
the  ingestion  of  large  amounts  of  sucrose,  lactose  or  glucose,  causing 
the  assimilation  limit  to  be  exceeded,  an  ahmentary  glycosuria  may 
arise.  In  diabetes  mellitus  very  large  amounts  of  glucose  are  excreted 
in  the  urine.  The  following  structural  formula  has  been  suggested  by 
Victor  Meyer  for  (/-glucose: 

CHO 

H— C— OH 

HO— C— H 

I 
H— C— OH 

I 
H— C— OH 

CH2OH 

(For  further  discussion  of  glucose  see  section  on  Hexoses,  page  20.) 

Experiments  on  Glucose 

The  following  tests  are  made  on  glucose  as  a  typical  carbohydrate 
and  are  not  specific  for  this  sugar.  A  specific  test  for  glucose  is  the 
Phenylhydrazine  Reaction  (3)  in  the  absence  of  a  positive  Resorcinol- 
Hydrochloric  Acid  Reaction  (see  page  35). 

1.  Solubility. — Test  the  solubility  of  glucose  in  the  "ordinary  solvents"  and 
in  alcohol.  (In  the  solubility  test  throughout  the  book  we  shall  designate  the 
following  solvents  as  the  "ordinary  solvents:"  H2O;  10  per  cent  NaCl;  0.5  per 
cent  Na^COs;  0.2  per  cent  HCl;  concentrated  KOH;  concentrated  HCl. ' 

2.  a-Naphthol  Reaction  (Mohsch).  Place  approximately  5  c.c.  of  concen- 
trated HoSO.)  in  a  test-tube.  Incline  the  tube  and  slowly  pour  down  the  inner 
side  of  it  approximately  5  c.c.  of  the  sugar  solution  to  which  2  drops  of  Mo- 
lisch's  reagent  (a  15  per  cent  alcoholic  solution  of  a-naphthol)  has  been  added, 
so  that  the  sugar  solution  will  not  mix  with  the  acid.  A  reddish-violet  zone 
is  produced  at  the  point  of  contact. 

'  See  Folin's  test  for  sugar  in  normal  urine  [Jour.  Biol.  Cheni.,  22,  327,  1915). 


22  PHYSIOLOGICAL    CHEMISTRY 

The  reaction  is  due  to  the  formation  of  furfural.^ 

HC— CH 

il      II 
HC     CCHO, 

\/ 

o 


by  the  acid.  The  test  is  given  by  all  bodies  containing  a  carbohydrate 
group  and  is  therefore  not  specific  and,  in  consequence,  of  very  little 
practical  importance. 

3.  Phenylhydrazine  Reaction. — Test  according  to  one  of  the  following 
methods :  (a)  To  a  small  amoimt  of  phenylhydrazine  mixture  (enough  to  fill  the 
rounded  portion  of  a  small  test-tube)  fiunished  by  the  instructor, ^  add  5  c.c. 
of  the  sugar  solution,  shake  well  and  heat  on  a  boiling  water-bath  for  one -half  to 
three-quarters  of  an  hour.  Allow  the  tube  to  cool  slowly  (not  under  the  tap) 
and  examine  the  crystals  microscopically  (Plate  III,  opposite) . 

If  the  solution  has  become  too  concentrated  in  the  boiling  process  it 
will  be  light  red  in  color  and  no  crystals  will  separate  until  it  is  diluted 
with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar  giving 
rise  to  an  osazone  of  a  definite  crystalline  form  which  is  typical  for  that 
sugar.  It  is  important  to  remember  in  this  connection  that  of  the 
simple  sugars  of  interest  in  physiological  chemistry,  glucose  and  fructose 
yield  the  same  osazone.  Each  osazone  has  a  definite  melting-point 
and  as  a  further  and  more  accurate  means  of  identification  it  may  be 
recrystallized  and  identified  by  the  determination  of  its  melting-point 
and  nitrogen  content.  The  reaction  taking  place  in  the  formation  of 
phenylglucosazone  is  as  follows : 


CH2OH 

I 
(CHOH)3 

CHOH 


+C6H5NH-NH2 


CHoOH 

(CH0H)3 
I  +C6H5NH-NH2- 

CHOH 
I  ^N-NHCeHs+HzO 


\H 

Glucose 


Phenylhydrazine 


\H 

Phenylhyd  razone 


'  According  to  v.  Ekenstein  and  Blanksma  {Ber.  d.  d.  chem.  GeselL,  43,  2358,  1910), 
oxymethylfurfural  is  formed. 

2  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazine  hydrochloride  and 
two  parts  of  sodium  acetate  by  weight.     These  are  thoroughly  mixed  in  a  mortar. 


PLATE  III. 


OSAZONES. 

Upper  form,  dextrosazone;  central  form,  maltosazone;  lower  form,  lactosazone. 


CARBOHYDRATES  23 

CH2OH  CH2OH 

(CH0H)3  (CH0H)3 

I  +C6H5NH-XH2^  I 

C  =  0  C  =  N-NHC6H5+H20 
I  ^N-NHC6H5+C6H5NH2+NH3  ^X-NHCsHb 

C  C 

Aniline  Glucosazone 

(b)  Place  5  c.c.  of  the  sugar  solution  in  a  test-tube,  add  i  c.c.  of  the  phenyl- 
hydrazine-acetate  solution  furnished  by  the  instructor/  and  heat  on  a  boiling 
water-bath  for  one -half  to  three-quarters  of  an  hour.  Allow  the  Uquid  to  cool 
slowly  and  examine  the  crystals  microscopically  (Plate  III,  opposite  p.  22). 

The  phenylhydrazine  test  has  been  so  modified  by  Cipollina  as  to 
be  of  use  as  a  rapid  clinical  test.  The  directions  for  this  test  are  given 
in  the  next  experiment. 

4.  Cipollina's  Test. — Thoroughly  mix  4  c.c.  of  dextrose  solution,  5  drops  of 
phenylhydrazine  (the  base)  and  J^  c.c.  of  glacial  acetic  acid  in  a  test-tube.  Heat 
the  mixture  for  about  one  minute  over  a  low  flame,  shaking  the  tube  continually  to 
prevent  loss  of  fluid  by  bumping.  Add  4-5  drogs  of  sodium  hydroxide  (sp.  gr.  1.16), 
being  certain  that  the  fluid  in  the  test-tube  remains  acid,  heat  the  mixture  again  for 
a  moment  and  then  cool  the  contents  of  the  tube.  Ordinarily  the  crystals  form  at 
once,  especially  if  the  sugar  solution  possesses  a  low  specific  gravity.  If  they  do 
not  appear  immediately  allow  the  tube  to  stand  at  least  20  minutes  before  deciding 
upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare  them  with  those  shown 
in  Plate  III,  opposite  page  22. 

5.  Riegler's  Reaction. ^ — Introduce  o.i  gram  of  phenylhydrazine-hydrochloride 
and  0.25  gram  of  sodium  acetate  into  a  test-tube,  add  20  drops  of  the  solution  under 
examination  and  heat  the  mixture  to  boiling.  Now  introduce  10  c.c.  of  a  3  per  cent 
solution  of  potassium  hydroxide  and  gently  shake  the  tube  and  contents.  If  the 
solution  under  e.Kamination  contains  dextrose  the  liquid  in  the  tube  will  assume  a 
red  color. 

One  per  cent  dextrose  yields  an  immediate  color,  whereas  0.05  per  cent  yields 
the  color  only  after  the  lapse  of  a  period  of  one-half  hour  from  the  time  the  alkali  is 
added.  In  urinary  examination  if  the  color  appears  after  the  thirty-minute  interval 
the  color  change  is  without  significance,  inasmuch  as  sugar-free  urine  will  respond 
thus.  The  reaction  is  given  by  all  aldehydes  and  therefore  the  test  cannot  be  safely 
employed  in  testing  urines  preserved  by  formaldehyde.  Albumin  does  not  interfere 
with  the  test. 

6.  Bottu's  Test. 3— To  8  c.c.  of  Bottu's  reagent^  in  a  test-tube  add  i  c.c.  of  the 

'  This  solution  is  prepared  by  mixlDg  one  part  by  volume,  in  each  case,  of  glacial  acetic 
acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the  base). 

^  Riegler:  Compl.  rend.  soc.  biol.,  66,  p.  795. 

'  Bottu:  Compt.  rend.  soc.  biol.,  66,  p.  972. 

*  This  reagent  contains  3.5  grams  of  o-nitrophenylpropiolic  acid  and  5  c.c.  of  a  freshly 
prepared  10  per  cent  solution  of  sodium  hydroxide  per  liter. 


24 


PHYSIOLOGICAL    CHEMISTRY 


solution  under  examination  and  mix  the  liquids  by  gentle  shaking.  Now  heat  the 
upper  portion  of  the  mixture  to  boiling,  add  an  additional  i  c.c.  of  the  solution  and 
heat  the  mixture  again  immediately.  The  appearance  of  a  blue  color  accompanied 
by  the  precipitation  of  small  particles  of  indigo  blue  indicates  the  presence  of  dex- 
trose in  the  solution  under  examination.  The  test  will  serve  to  detect  the  presence 
of  O.I  per  cent  of  dextrose. 

7.  Precipitation  by  Alcohol. — To  10  c.c.  of  05  per  cent  alcohol  add  about  2  c.c. 
of  dextrose  solution.  Compare  the  result  with  that  obtained  under  Dextrin,  5, 
page  48. 

8.  Iodine  Test. — Make  the  regular  iodine  test  as  given  under  Starch,  5,  page  45, 
and  keep  this  result  in  mind  for  comparison  with  the  results  obtained  later  with 
starch  and  with  dextrin. 

9.  Diffusibility  of  Glucose. — Test  the  diffusibility  of  glucose  solution  through 
animal  membrane  or  parchment  paper,  making  a  dialyzer  hke  one  of  the  models 
shown  in  Fig.  2. 


Fig.  2. — DiALYziNG  Apparatus  for  Students'  Use. 


A  most  satisfactory  dialyzing  bag  may  be  made  of  collodion  as  follows: 
Pour  a  solution  of  collodion  into  a  clean  dry  Erlenmeyer  flask  or  test-tube. 
While  rotating  the  vessel  on  its  longitudinal  axis,  gradually  pour  out  the  collodion, 
at  the  same  time  being  careful  that  the  interior  surface  of  the  flask  is  completely 
coated  with  the  solution.  Continue  the  rotation  in  the  inverted  position  until 
the  collodion  ceases  to  flow.  After  the  solution  has  evaporated  such  that  the 
collodion  skin  on  the  rim  is  dry  and  stiff,  cut  or  loosen  it  around  the  edge  of  the 
rim.  With  a  pipette  or  wash  bottle  run  in  a  few  cubic  centimeters  of  water  be- 
tween the  membrane  and  the  wall  of  the  flask  or  test-tube.  Shake  the  incUned 
vessel  while  rotating  on  its  longitudinal  axis,  thus  detaching  the  membrane. 
Now  withdraw  the  detached  bag  and  fill  with  water,  to  determine  whether  or  not 
it  contains  defects.' 

All  monosaccharides  and  disaccharides  are  diffusible,  but  many  polysac- 
charides are  not. 

10.  Moore's  Test. — To  2-3  c.c.  of  sugar  solution  in  a  test-tube  add  an  equal 
volume  of  concentrated  KOH  or  NaOH,  and  boil.  The  solution  darkens  and  finally 
assumes  a  brown  color.  .  At  this  point  the  odor  of  caramel  may  be  detected. 

This  is  an  exceedingly  crude  test  and  is  of  little  practical  value.  The  alkali 
brings  about  condensation  and  decomposition.  The  brown  color  is  due  to  the 
formation  of  condensation  products.  Among  the  decomposition  products  are  the 
potassium  or  sodium  salts  of  certain  organic  acids. 

^  Gies:  Quoted  by  Clark.     Bioch.  Bull.,  i,  198,  1911. 


CARBOHYDRATES  25 

II.  Reduction  Tests. — To  their  aldehyde  or  ketone  structure  many 
sugars  owe  the  property  of  readily  reducing  alkaline  solutions  of  the 
oxides  of  metals  like  copper,  bismuth  and  mercury;  they  also  possess 
the  property  of  reducing  ammoniacal  silver  solutions  with  the  separa- 
tion of  metallic  silver.  Upon  this  property  of  reduction  the  most 
widely  used  tests  for  sugars  are  based.  When  whitish-blue  cupric 
hydroxide  in  suspension  in  an  alkaline  liquid  is  heated  it  is  converted 
into  insoluble  black  cupric  oxide,  but  if  a  reducing  agent  like  certain 
sugars  be  present  the  cupric  hydroxide  is  reduced  to  insoluble  yellow 
cuprous  hydroxide,  which  in  turn,  on  further  heating,  may  be  converted 
into  brownish-red  or  red  cuprous  oxide.  These  changes  are  indicated 
as  follows: 

OH 

/ 
Cu  ^Cu  =  0+H20. 

\  Cupric  o-Tide 

\  (black). 

OH 

Cupric  hydroxide 
(whitish-blue). 


Cu 


OH 


OH 


OH 


-^  2Cu-OH-t-H20+0. 

Cuprous  hydroxide 
(yellow). 


Cu 


OH 

Cu 

Cu— OH 

\ 

1                    -s 

0-hHoO 

Cu— OH 

/ 

Cu 

Cuprous  hydroxide 
(yellow). 

Cuprous    oxide 
(brownish-red) . 

The  chemical  equations  here  discussed  are  exemplified  in  Trommer's 
and  Fehling's  tests. 

(c)  Trommer's  Test. — To  5  c.c.  of  sugar  solution  in  a  test-tube  add  one-half  it.s 
volume  of  KOH  or  NaOH.  Mix  thoroughly  and  add,  drop  by  drop,  a  very  dilute 
solution  of  copper  sulphate.  Continue  the  addition  until  there  is  a  slight  permanent 
precipitate  of  cupric  hydroxide  and  in  consequence  the  solution  is  slightly  turbid. 
Heat,  and  the  cupric  hydroxide  is  reduced  to  yellow  cuprous  hydroxide  or  to  brown- 
ish-red cuprous  o.xide. 

If  the  solution  of  copper  sulphate  used  is  too  strong  a  small  brownish-red  pre- 
cipitate produced  in  a  weak  sugar  solution  may  be  entirely  masked.     On  the  other 


26  PHYSIOLOGICAL   CHEMISTRY 

hand,  particularly  in  testing  for  sugar  in  the  urine,  if  too  little  copper  sulphate  is 
used  a  light-colored  precipitate  formed  by  uric  acid  and  purine  bases  may  obscure 
the  brownish-red  precipitate  of  cuprous  oxide.  The  action  of  KOH  or  NaOH  in  the 
presence  of  an  excess  of  sugar  and  insufficient  copper  will  produce  a  brownish  color. 
Phosphates  of  the  alkaline  earths  may  also  be  precipitated  in  the  alkaline  solution 
and  be  mistaken  for  cuprous  hydroxide.     Trommer's  test  is  not  very  satisfactory. 

Salkowski^  has  proposed  a  modification  of  the  Trommer  procedure  which  he 
claims  is  a  very  accurate  sugar  test. 

(b)  Fehling's  Test. — To  about  i  c.c.  of  Fehling's  solution^  in  a  test-tube  add 
about  4  c.c.  of  water,  and  boil.^  [The  cupric  hydroxide  is  held  in  solution  by  the 
sodium  potassium  tartrate  (Rochelle  salt).]  This  is  done  to  determine  whether 
the  solution  will  of  itself  cause  the  formation  of  a  precipitate  of  brownish-red 
cuprous  oxide.  If  such  a  precipitate  forms,  the  Fehling's  solution  must  not  be 
used.  Add  sugar  solution  to  the  warm  Fehling's  solution  a  few  drops  at  a  time 
and  heat  the  mixture  after  each  addition.  The  production  of  yellow  cuprous  hy- 
droxide or  brownish-red  cuprous  oxide  indicates  that  reduction  has  taken  place. 
The  yellowish  precipitate  is  more  likely  to  occur  if  the  sugar  solution  is  added 
rapidly  and  in  large  amount,  whereas  with  a  less  rapid  addition  of  smaller 
amounts  of  sugar  solution  the  brownish-red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even  this 
test  is  not  entirely  reliable  when  tfsed  to  detect  sugar  in  the  urine.  Such 
bodies  as  conjugate  glycuronates,  uric  acid,  nucleoprotein  and  homogen- 
tisic  acid  when  present  in  sufficient  amount  may  produce  a  result  simi- 
lar to  that  produced  by  sugar.  Phosphates  of  the  alkaline  earths  may 
be  precipitated  by  the  alkali  of  the  Fehling's  solution  and  in  appearance 
may  be  mistaken  for  cuprous  hydroxide.  Cupric  hydroxide  may  also 
be  reduced  to  cuprous  oxide  and  this  in  turn  be  dissolved  by  creatinine, 
a  normal  urinary  constituent.  This  will  give  the  urine  under  examina- 
tion a  greenish  tinge  and  may  obscure  the  sugar  reaction  even  when  a 
considerable  amount  of  sugar  is  present.  According  to  Laird, "*  even 
small  amounts  of  creatinine  will  retard  the  reaction  velocity  of  reducing 
sugars  with  Fehling's  solution. 

In  testing  urine  preserved  by  chloroform  a  positive  test  may  be  ob- 
tained in  the  absence  of  sugar.  This  is  due  to  the  fact  that  the  hot 
alkali  produces  formic  acid  (a  reducing  fatty  acid)  from  the  chloroform. 

'  Salkowski:  Zeit.  physiol.  Chem.,  79,  164,  1912. 

2  Fehling's  solution  is  composed  of  two  definite  solutions — a  copper  sulphate  solution 
and  an  alkaline  tartrate  solution — which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =  54.6$  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =  i2S  grams  of  potassium  hydroxide  and  173  grams  of  Rochelle 
salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

*  More  dilute  Fehling's  solution  should  be  used  in  testing  very  dilute  sugar  solutions. 
In  case  of  concentrated  sugar  solutions  it  may  sometimes  be  desirable  to  use  a  larger  volume 
of  the  Fehling's  solution. 

''  Laird:  Journal  of  Pathology  and  Bacteriology,  16,  398,  1912. 


CARBOHYDRATES  2  7 

Ammonium  salts  also  interfere  with  Fehling's  test.  If  present  in 
excess  the  solution  {e.g.,  urine)  should  be  made  alkaline  and  boiled  in 
order  to  decompose  the  ammonium  salts. 

If  the  solution  under  examination  by  Fehling's  test  is  acid  in  re- 
action it  must  be  neutralized  or  made  alkaline  before  applying  the  test. 

(c)  Benedict's  Modifications  of  Fehling's  Test. — First  Modification. — To  2  c.c. 
of  Benedict's  solution'  in  a  test-tube  add  6  c.c.  of  distilled  water  and  79  drops 
(not  morej  of  the  solution  under  examination.  Boil  the  mixture  vigorously  for 
about  15-30  seconds  and  permit  it  to  cool  to  room  temperature  spontaneously. 

If  sugar  is  present  in  the  solution  a  precipitate  will  form  which  is 
often  bluish-green  or  green  at  first,  especially  if  the  percentage  of  sugar  is 
low,  and  which  usually  becomes  yellowish  upon  standing.  If  the  sugar 
present  exceeds  0.06  per  cent  this  precipitate  generally  forms  at  or  below 
the  boiling-point,  whereas  if  less  than  0.06  per  cent  of  sugar  is  present  the 
precipitate  forms  more  slowly  and  generally  only  after  the  solution  has 
cooled. 

Benedict  claims,  whereas  the  original  Fehling  test  will  not  serve 
to  detect  sugar  when  present  in  a  concentration  of  less  than  o.i  per 
cent,  that  the  above  modification  will  serve  to  detect  sugar  when 
present  in  as  small  quantity  as  0.015-0.02  per  cent.  Corroboration 
of  this  claim  of  increased  delicacy  has  recently  been  submitted  by 
Harrison. - 

The  modified  Fehling  solution  used  in  the  above  test  differs  from 
the  original  Fehling  solution  in  that  100  grams  of  sodium  carbonate  is 
substituted  for  the  125  grams  of  potassium  hydroxide  ordinarily  used, 
thus  forming  a  Fehling  solution  which  is  considerably  less  alkaline 
than  the  original.  This  alteration  in  the  composition  of  the  Fehling 
solution  is  of  advantage  in  the  detection  of  sugar  in  the  urine  inasmuch 
as  the  strong  alkalinity  of  the  ordinary  Fehling  solution  has  a  tendency, 
when  the  reagent  is  boiled  with  a  urine  containing  a  small  amount  of 
dextrose,  to  decompose  sufficient  of  the  sugar  to  render  the  detection  of 
the  remaining  portion  exceedingly  difficult  by  the  usual  technic.  Bene- 
dict claims  that  for  this  reason  the  use  of  this  modified  solution  permits 
the  detection  of  much  smaller  amounts  of  sugar  than  does  the  use  of  the 
ordinarily  Fehling  solution. 

'  Benedict's  modified  Fehling  solution  consists  of  two  definite  solutions — a  copper  sul- 
phate solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =  24-(>5  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =  ioo  grams  of  anhydrous  sodium  carbonate  and  173  grams  of 
Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mi.xed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

*  Harrison:  Fliarm.  Jour.,  87,  746,  1911. 


28  PHYSIOLOGICAL    CHEMISTRY 

Second  Modification.  ^ — Benedict  has  fvirther  modified  his  solution  and  has 
succeeded  in  obtaining  one  which  does  not  deteriorate  upon  long  standing.  ^ 
The  following  is  the  procedure  for  the  detection  of  glucose  in  solution :  To  5  c.c. 
of  the  reagent  in  a  test-tube  add  8  (not  more)  drops  of  the  solution  under  exam- 
ination. Boil  the  mixture  vigorously  for  from  one  to  two  minutes  and  then  allow 
the  fluid  to  cool  spontaneously.  In  the  presence  of  dextrose  the  entire  body  of 
the  solution  will  be  filled  with  a  precipitate,  which  may  be  red,  yellow  or  green  in 
color,  depending  upon  the  amount  of  sugar  present.  If  no  glucose  is  present,  the 
solution  will  remain  perfectly  clear.  (If  urine  is  being  tested,  it  may  show  a  very 
faint  turbidity,  due  to  precipitated  urates.) 

Even  very  small  quantities  of  glucose  (o.i  per  cent)  yield  precipitates 
of  surprising  bulk  with  this  reagent,  and  the  positive  reaction  for  glucose 
is  the  filling  of  the  entire  body  of  the  solution  with  a  precipitate,  so  that 
the  solution  becomes  opaque.  Since  amount  rather  than  color  of  the 
precipitate  is  made  the  basis  of  this  test,  it  may  be  applied  even  for  the 
detection  of  small  quantities  of  glucose,  as  readily  in  artificial  light  as  in 
daylight.  Chloroform  does  not  interfere  with  this  test  nor  do  uric  acid 
or  creatinine  interfere  to  such  an  extent  as  in  the  case  of  Fehling's  test. 

Mercuric  Oxide  Reduction  Test  (Cramer).^ — This  test  depends  on 
the  reduction  of  mercuric  oxide  in  a  weakly  alkaline  solution,  with  the 
formation  of  metallic  mercury.  The  degree  of  alkalinity  is  an  important 
factor,  as  the  test  becomes  more  sensitive  but  less  specific  the  greater 
the  alkalinity  of  the  reagent. 

For  the  detection  of  reducing  sugar  in  aqueous  solution  proceed  as  follows : 
Introduce  3  c.c.  of  Cramer's  "2.5  reagent"  into  a  test-tube^  and  heat  to  boiling. 
The  solution  remains  clear,  but  turns  sUghtly  yellow.  Add  3  c.c.  of  the  sugar 
solution  and  again  heat  to  boiling.  Remove  the  tube  from  the  flame  and  note 
that  the  mixture  becomes  turbid,  darkens,  and  that  on  standing  a  precipitate 
of  finely  divided  mercury  settles  to  the  bottom  of  the  tube. 

^  Benedict:  Jour.  Am.  Med.  Ass'n,  57,  1193,  1911. 

^  Benedict's  new  solution  has  the  following  composition: 

Copper  sulphate i7-3  grams. 

Sodium  citrate 1730  grams. 

Sodium  carbonate  (anhydrous) 100. o  grams. 

Distilled  water  to  make  i  liter. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600  c.c  of  water. 
Pour  (through  a  folded  filter  paper  if  necessary)  into  a  glass  graduate  and  make  up  to  850  c.c. 
Dissolve  the  copper  sulphate  in  about  100  c.c.  of  water  and  make  up  to  150  c.c.  Pour  the 
carbonate-citrate  solution  into  a  large  beaker  or  casserole  and  add  the  copper  sulphate  solu- 
tion slowly,  with  constant  stirring.  The  mixed  solution  is  ready  for  use  and  does  not  dete- 
riorate upon  long  standing. 

^  Cramer:  Bioch.  Jour.,  9,  156,  1915. 

*  Cramer's  "2.5  Reagent." — 0.4  gram  mercuric  oxide  (red  or  yellow)  and  6  grams  potas- 
sium iodide  are  dissolved  in  100  c.c.  water.  This  solution  is  weakly  alkaline.  The  alka- 
linity must  now  be  adjusted  so  that  10  c.c.  of  the  reagent  are  neutralized  by  2.5  c.c.  of 
N/io  acid,  using  phenolphthalein  as  indicator.  This  is  done  by  titrating  10  c.c.  of  the 
reagent  with  X/io  acid  and,  after  the  alkalinity  of  the  reagent  has  thus  been  determined, 
adding  the  requisite  amount  of  X/io  acid  or  alkali  to  the  bulk  of  the  reagent.  The  reagent 
is  a  clear  colorless  solution  which  turns  slightly  yellow  on  heating  and  becomes  colorless 
again  on  cooling.     It  must  rernain  clear  on  boiling. 


CARBOHYDRATES  29 

This  test  is  positive  with  such  sugars  as  give  other  reduction  tests, 
e.g.,  glucose,  fructose,  lactose,  maltose,  arabinose,  etc.  If  the  reducing 
sugar  be  present  in  aqueous  solution  a  slight  reduction  may  be  obtained 
with  I  mg.  of  glucose.  If  the  reagent  be  made  strongly  alkaline,  it 
ceases  to  be  specific  for  reducing  sugars  and  chemically  allied  sub- 
stances and  is  reduced  by  other  organic  substances,  e.g.,  creatinine. 

It  is  claimed  that  this  test  is  free  from  fallacies  inherent  in  Fehling's 
test  as  the  result  of  the  reducing  action  of  uric  acid  and  creatinine. 

The  test  is  more  sensitive  than  the  Fehling's  or  Xylander's  reac- 
tions, and  is  particularly  suitable  for  the  examination  of  urines  in  which 
the  amount  of  sugar  present  exceeds  the  normal  amount  only  slightly. 

(e)  Bismuth  Reduction  Test  (Boettger). — To  5  c.c.  of  sugar  solution  in  a  test-tube 
add  I  c.c.  of  KOH  or  NaOH  and  a  very  small  amount  of  bismuth  subnitrate,  and 
boil.  The  solution  will  gradually  darken  and  finally  assume  a  black  color  due  to 
reduced  bismuth.  If  the  test  is  made  on  urine  containing  albumin  this  must  be 
removed,  by  boiling  and  filtering,  before  applying  the  test,  since  with  albumin  a 
similar  change  of  color  is  produced  (bismuth  sulphide). 

(f)  Bismuth  Reduction  Test  (Nylander). — To  5  c.c.  of  sugar  solution  in  a  test- 
tube  add  one-tenth  its  volume  of  Nylander's  reagent'  and  heat  for  fi.ve  minutes 
in  a  boiling  water-bath.-  The  solution  will  darken  if  reducing  sugar  is  present, 
and  upon  standing  for  a  few  moments  a  black  color  wiU  appear. 

This  color  is  due  to  the  precipitation  of  bismuth.  If  the  testis  made 
on  urine  containing  albumin  this  must  be  removed,  by  boiling  and 
filtering,  before  applying  the  test,  since  with  albumin  a  similar  change  of 
color  is  produced.  Glucose  when  present  to  the  extent  of  0.08  per  cent 
may  be  easily  detected  by  this  reaction  (Rabe^  claims  that  o.oi  per  cent 
sugar  may  be  so  detected).  Uric  acid  and  creatinine  which  interfere 
with  the  Fehling's  test  do  not  interfere  with  the  Xylander  test.  It 
is  claimed  by  Bechold  that  the  bismuth  reduction  tests  give  a  nega- 
tive reaction  \vith  solutions  containing  sugar  when  mercuric  chloride  or 
chloroform  is  present.  Other  observers^  have  failed  to  verify  the 
inhibitory  action  of  mercuric  chloride  and  have  shown  that  the  in- 
hibitory influence  of  chloroform  may  be  overcome  by  raising  the  tem- 
perature of  the  urine  to  the  boiling-point  for  a  period  of  live  minutes 
previous  to  making  the  test.  Urines  rich  in  indican.  urochromc,  urocry- 
thrin  or  hemato porphyrin,  as  well  as  urines  excreted  after  the  ingestion  of 
large  amounts  of  certain  medicinal  substances,  may  give  a  darkening  of 

^  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  subnitrate  and  4  grams 
of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium  hydroxide  solution.  The  reagent  is 
then  cooled  and  filtered. 

-  Hammarsten  suggests  that  the  mi.xture  should  be  boiled  2-5  minutes  (according  to  the 
sugar  content)  over  a  free  flame  and  the  tube  then  permitted  to  stand  5  minutes  before 
drawing  conclusions. 

^Rabe:  Apotlt.  Ztg.,  29,  554,  1Q14. 

^  Rehfusp  and  Hawk:  Journal  of  Biological  Chemistry;  7,  267,  igio;  also  Zeidlitz: 
U psala  Lakarcforen  Fork.,  N.  F.,  ii,  1906. 


30  PHYSIOLOGICAL   CHEMISTRY 

Nylander's  reagent  similar  to  that  of  a  true  sugar  reaction.  It  is  a  dis- 
puted point  whether  the  urine  after  the  administration  of  urotropin 
will  reduce  Nylander's  reagent.^  Strausz^  has  recently  shown  that  the 
urine  of  diabetics  to  whom  "lothion"  (diiodohydroxypropane)  has 
been  administered  will  give  a  negative  Nylander-Almen  reaction  and 
respond  positively  to  the  Fehling  and  polariscopic  tests.  "lothion" 
also  interferes  with  the  Nylander-Almen  test  in  vitro  whereas  KI  and 
I  do  not. 

According  to  Rustin  and  Otto,  the  addition  of  PtCU  increases  the 
delicacy  of  Nylander-Almen  reaction.  Theyclaim  that  this  procedure 
causes  the  sugar  to  be  converted  quantitatively.  No  quantitative 
method  has  yet  been  devised,  however,  based  upon  this  principle. 

Bohmansson^  before  testing  the  urine  under  examination  treats 
it  (lo  c.c.)  with  J-^  volume  of  25  per  cent  hydrochloric  acid  and  about 
}4  volume  of  boneblack.  This  mixture  is  shaken  one  minute,  then 
filtered  and  the  neutralized  filtrate  tested  by  Nylander-Almen  reaction. 
Bohmansson  claims  that  this  procedure  removes  certain  interfering 
substances,  in  particular  urochrome. 

A  positive  bismuth  reduction  test  is  probably  due  to  the  following 
reactions: 

(a)  Bi(OH)2N03  +  K0H-^Bi(0H)3  +  KNO3. 

(b)  2Bi(OH)3— 30->Bi2  +  3H2O. 

(g).  Barfoed's  Test. — Place  about  5  c.c.  of  Barfoed's  solution*  in  a  test-tube 
and  heat  to  boiling.  Add  glucose  solution  slowly,  a  few  drops  at  a  time,  heating 
after  each  addition.  Reduction  is  indicated  by  the  formation  of  a  red  precipitate 
of  cuprous  oxide.  If  the  precipitate  does  not  form  upon  continued  boiling  allow 
the  tube  to  stand  a  few  minutes  and  examine. 

According  to  Welker^  chlorides  interfere  pronouncedly  with  the 
reaction  causing  the  formation  of  a  green  precipitate. 

Barfoed's  test  is  not  a  specific  test  for  glucose  as  is  frequently  stated, 
but  simply  serves  to  detect  monosaccharides.  Disaccharides  will  also 
respond  to  the  test,  under  proper  conditions  of  acidity.^  Also  if  the 
sugar  solution  is  boiled  sufficiently  long,  in  contact  with  the  reagent,  to 
hydrolyze  the  disaccharide  through  the  action  of  the  acetic  acid  present 
in  the  Barfoed's  solution  a  positive  test  results.''     Barfoed's  is  a  copper 

^Abt:  Archives  of  Pediatrics,  24,  275,  1907;  also  Weitbrecht:  Schiveiz.  Wochschr.,  47, 
577.  1909- 

*  Strausz:  Munch,  med.  Woch.,  59,  85, 191 2. 
'  Bohmansson:  Biochem.  Zeit.,  19,  p.  281. 

*  Barfoed's  solution  is  prepared  as  follows:  Dissolve  4.5  grams  of  neutral  crystallized 
copper  acetate  in  100  c.c.  of  water  and  add  1.2  c.c.  of  50  per  cent  acetic  acid.  This  solu- 
tion should  be  freshly  made. 

*  Welker:  Jour.  Am.  Chem.  Soc,  37,  2227,  1915. 

*  Mathews  and  McGuigan:  Am.  Jour.  Physiol.,  19,  175,  1907. 

'  Hinkle  and  Sherman:  Jour.  Am.  Chem.  Soc,  29,  1744,  1907. 


CARBOHYDRATES  3 1 

reduction  test,  but  differs  from  Fehling's  and  other  reduction  tests  in 
that  the  reduction  is  brought  about  in  an  acid  solution.  It  is  unsuited 
for  the  detection  of  sugar  in  urine. 

12.  Fermentation  Test. — "Rub  up"  in  a  mortar  about  20  c.c.  of  the  sugar  solu- 
tion with  a  small  piece  of  compressed  yeast.  Transfer  the  mixture  to  a  sacchar- 
ometer  (shown  in  Fig.  3)  and  stand  it  aside  in  a  warm  place  for  about  twelve 
hours.  If  the  sugar  is  fermentable,  alcoholic  fermentation  will  occur  and  carbon 
dioxide  will  collect  as  a  gas  in  the  upper  portion  of  the  tube  (see  Fig.  4;. 
On  the  completion  of  fermentation  introduce  a  Uttle  potassium  hydroxide  solu- 
tion into  the  graduated  portion  by  means  of  a  bent  pipette,  place  the  thumb 
tightly  over  the  opening  in  the  apparatus  and  invert  the  saccharometer.  Re- 
membering that  KOH  has  the  power  to  absorb 
CO 2  how  do  you  explain  the  result?' 

13.  Formation  of  Caramel. — Gently  heat  a 
small  amount  of  pulverized  dextrose  in  a  test-tube. 
After  the  sugar  has  melted  and  turned  brown,  allow 
the  tube  to  cool,  add  water  and  warm.  The  color- 
ing matter  produced  is  known  as  caramel. 


s^Vk 


Fig.  3. — EixHORN  Saccharometer. 


Fig.  4. — Illustrating  Different  Stages 
IN  Fermentation. 


14.  Demonstration  of  Optical  Activity. — A  demonstration  of  the  use  of  the 
polariscope,  by  the  instructor,  each  student  being  required  to  take  readings  and 
compute  the  "specific  rotation." 


Use  of  the  Polariscope 

For  a  detailed  description  of  the  different  forms  of  polariscopes,  the 
method  of  manipulation  and  the  principles  involved,  the  student  is 
referred  to  any  standard  text-book  of  physics.  A  brief  description  fol- 
lov^^s:     An  ordinary  ray  of  light  vibrates  in  every  direction.     If  such  a 

1  The  findings  of  Neuberg  and  associates-  indicate  that  the  liberation  of  carbon  di- 
oxide by  yeast  is  not  necessarily  a  criterion  of  the  presence  of  sugar.  The  presence  of 
an  enzyme  called  carftovy/fl^e  has  been  demonstrated  in  yeast  which  has  the  power  of 
splitting  off  C02  from  the  carboxyl  group  of  amino- and  other  aliphatic  acids. 

^  Neuberg  and  .Associates:  Biochcm.  Zcitscli.,  31,  170;  32,  T,2y,  36,  (60,  68,  76),  1911. 


32 


PHYSIOLOGICAL   CHEMISTRY 


ray  is  caused  to  pass  through  a  "polarizing"  Nicol  prism  it  is  resolved 
into  tivo  rays,  one  of  which  \dbrates  in  every  direction  as  before  and  a 
second  ray  which  vibrates  in  one  plane  only.  This  latter  ray  is  said  to 
be  polarized.  Many  organic  substances  (sugar,  proteins,  etc.)  have  the 
power  of  twisting  or  rotating  this  plane  of  polarized  light,  the  extent  to 
which  the  plane  is  rotated  depending  upon  the  number  of  molecules 
which  the  polarized  light  passes.  Substances  which  possess  this  power 
are  said  to  be  "optically  active."  The  specific  rotation  of  a  substance  is 
the  rotation  expressed  in  degrees  which  is  afforded  by  i  gram  of  sub- 


FiG.  5- — One  Form  or  Laurent  Polariscope. 

B,  ^Microscope  for  reading  the  scale;  C,  a  vernier;  E,  position  of  the  analyzing  Nicol  prism; 
H,  polarizing  Nicol  prism  in  the  tube  below  this  point. 

Stance  dissolved  in  i  c.c.  of  water  in  a  tube  one  decimeter  in  length. 
The  specific  rotation,  (Q:)n,  may  be  calculated  by  means  of  the  following 
formula: 


Wz,= 


p.i' 


in  which 


J)  =  sodium  light. 
a  =  observed  rotation  in  degrees. 
p  —  grams  of  substance  dissolved  in  i  c.c.  of  liquid. 
/  =  length  of  the  tube  in  decimeters. 

If  the  specific  rotation  has  been  determined  and  it  is  desired  to  ascer- 
tain the  per  cent  of  the  substance  in  solution,  this  may  be  obtained  by 
the  use  of  the  following  formula, 


P  = 


{a)D  I 


The  value  of  p  multiplied  by  loo  will  be  the  percentage  of  the  sub- 
stance in  solution. 


CARBOHYDRATES  33 

SPECIFIC  ROTATIONS  OF  MORE  COMMON  CARBOHYDRATES  i 

</-Glucose +  52.5°      Sucrose +  66.5' 

(i-Fructose —  92.3°      Lactose +  52.5° 

<f-Galactose +81.5°      Maltose +137.0° 

£j-Mannose +14.2°      Raffinose +104.0" 

/-Arabinose +104.5°      Dextrin +195.0° 

/-Xylose +  19.0°      Starch  (soluble) +196.0° 

Rhamnose +     9.0°      Glycogen +197.0° 

An  instrument  by  means  of  which  the  extent  of  the  rotation  may  be 
determined  is  called  a  polariscope  or  polarimeter.  Such  an  instru- 
ment designed  especially  for  the  examination  of  sugar  solutions  is 
termed  a  saccharimeter  or  polarizing  saccharimeler.  The  form  of  polari- 
scope in  Fig.  5,  page  32,  consists  essentially  of  along  barrel  pro\-ided 
with  a  Nicol  prism  at  either  end  (Fig.  6,).  The  solution  under  exami- 
nation is  contained  in  a  tube  which  is  placed  between  these  two  prisms. 


Fig.  6. — Diagr.xmmatic  Representation  of  the  Course  of  the  Light  through  the 
Laurent  Polariscope.     (The  direction  is  reversed  from  that  of  Fig.  5,  p.  32.) 

a,  Bichromate  plate  to  purify  the  light;  h,  the  polarizing  Nicol  prism;  c,  a  thin  quartz 
plate  covering  one-half  the  field  and  essential  in  producing  a  second  polarized  plane;  d, 
tube  to  contain  the  liquid  under  examination;  e,  the  analyzing  Nicol  prism;  /and  g,  ocular 
lenses. 

At  the  front  end  of  the  instrument  is  an  adjusting  eyepiece  for  focusing 
and  a  large  recording  disc  which  registers  in  degrees  and  fractions  of  a 
degree.  The  light  is  admitted  into  the  far  end  of  the  instrument  and  is 
polarized  by  passing  through  a  Nicol  prism.  This  polarized  ray  then 
traverses  the  column  of  liquid  within  the  tube  mentioned  above  and 
if  the  substance  is  optically  active  the  plane  of  the  polarized  ray  is 
rotated  to  the  right  or  left.  Bodies  rotating  the  ray  to  the  right  are 
called  dextro-rotatory  and  those  rotating  it  to  the  left  levo-rotalorv. 

Within  the  apparatus  is  a  disc  which  is  so  arranged  as  to  be  without 
lines  and  uniformly  light  at  zero.  Upon  placing  the  optically  active 
substance  in  position,  however,  the  plane  of  polarized  light  is  rotated 
or  turned  and  it  is  necessary  to  rotate  the  disc  through  a  certain  number 
of  degree  in  order  to  secure  the  normal  conditions,  i.e.,  "without  lines 

1  The  specific  rotation  varies  with  the  temperature  and  concentration  of  the  solution. 
The  figures  here  given  are  for  concentrations  of  about  10  per  cent  and  temperatures  of  about 
20°C.  Fresh  solutions  may  give  markedly  different  values  due  to  mutarotation,  the  figures 
here  given  representing  the  constant  values  obtained  on  standing. 

3 


34 


PHYSIOLOGICAL   CHEMISTRY 


and  uniformly  light."     The  difference  between  this  reading  and  the 
zero  is  a  or  the  observed  rotation  in  degrees. 

Polarizing  saccharimeters  are  also  constructed  by  which  the  per- 
centage of  sugar  in  solution  is  determined  by  making  an  observation 
and  multiplying  the  value  of  each  division  on  a  horizontal  sliding  scale 
by  the  value  of  the  division  expressed  in  terms  of  dextrose.  This 
factor  may  vary  according  to  the  instrument. 


Fig.  7. — PoLAEiscoPE  (Schmidt  and  Hansch  Model). 

"Optical"  raethods  embracing  the  determination  of  the  optical 
activity  are  being  utilized  in  recent  years  in  many  "quantitative" 
connections.^ 

CH2OH 

FRUCTOSE   (CH0H)3 

CO 


CH20H 

'  Abderhalden  and  Schmidt:  "Determination  of  blood  content  by  means  of  the  optical 
method,"  Zeit.  physiol.  Chem.,  66,  120,  1910;  also  C.  Neuberg:  "Determination  of  nucleic 
acid  cleavage  by  polarization,"  Biochemische  Zeiischrift,  30,  505,  191 1. 


CARBOHYDRATES  35 

As  already  stated,  fructose,  sometimes  called  levulose  or  fruit  sugar, 
occurs  widely  disseminated  throughout  the  plant  kingdom  in  company 
with  glucose.  Its  reducing  power  is  somewhat  weaker  than  that  of 
dextrose.  Fructose  does  not  ordinarily  occur  in  the  urine  in  diabetes 
mellitus,  but  has  been  found  in  exceptional  cases.  With  phenylhydra- 
zine  it  forms  the  same  osazone  as  glucose.  With  methylphenyUiy- 
drazine,  levulose  forms  a  characteristic  methylphenylfructosazone. 

(For  a  further  discussion  of  fructose  see  the  section  on  Hexoses, 
page  20.) 

Experiments  on  Fructose 

1-6.  Repeat  Solubility,  Fehling's,  Phenylhydrazine,  Barfoed's,  Nylander's, 
and  Fermentation  tests  as  given  under  Glucose,  pages  21-31. 

7.  Resorcinol-Hydrochloric  Acid  Reaction  (Seliwanoff). — To  5  c.c.  of  Seli- 
wanoff's  reagent^  in  a  test-tube  add  a  few  drops  of  a  fructose  solution  and  heat  the 
mixture  to  boiling.  A  positive  reaction  is  indicated  by  the  production  of  a  red 
color  and  the  separation  of  a  brown-red  precipitate.  The  latter  may  be  dissolved 
in  alcohol  to  which  it  will  impart  a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained  with 
solutions  of  glucose  or  maltose.  This  has  been  explained-  in  the  case 
of  glucose  as  due  to  the  transformation  of  the  glucose  into  fructose 
by  the  catalytic  action  of  the  hydrochloric  acid.  The  precautions  neces- 
sary for  a  positive  test  for  levulose  are  as  follows:  The  concentration 
of  the  hydrochloric  acid  must  not  be  more  than  12  per  cent.  The  reac- 
tion (red  color)  and  the  precipitate  must  be  observed  after  not  more 
than  20-30  seconds  boiling.  Glucose  must  not  be  present  in  amounts 
exceeding  2  per  cent.  The  precipitate  must  be  soluble  in  alcohol  with 
a  bright  red  color. 

8.  Borchardt's  Reaction. — To  about  5  c.c.  of  a  solution  of  fructose  in  a  test- 
tube  add  an  equal  volume  of  25  per  cent  hydrochloric  acid  and  a  few  crystals  of 
resorcinol.  Heat  to  boiling  and  after  the  production  of  a  red  color,  cool  the  tube 
under  running  water  and  transfer  to  an  evaporating  dish  or  beaker.  Make  the 
mixture  slightly  alkaline  with  solid  potassium  hydroxide,  return  it  to  a  test-tube, 
add  2-3  c.c.  .of  acetic  ether  and  shake  the  tube  vigorously.  In  the  presence  of 
levulose,  the  acetic  ether  is  colored  yellow.  (For  further  discussion  of  the  test  see 
Chapter  XXIII.) 

9.  Formation  of  Methylphenylfructosazone. — To  a  solution  of  i.S  grams  of 
levulose  in  10  c.c.  of  water  add  4  grams'  of  methylphenylhydrazine  and  enough 
alcohol  to  clarify  the  solution.  Introduce  4  c.c.  of  50  per  cent  acetic  acid  and  heat 
the  mixture  for  5-10  minutes  on  a  boiling  water-bath.*     On  standing  15  minutes 

^  Seliwanoff's  reagent  may  be  prepared  by  dissolving  0.05  gram  of  resorcinol  in  100  c.c. 
of  dilute  (1:2)  hydrochloric  acid. 

*  Koenigsfeld:  Bioch.  Zcil.,  38,  311,  1912. 
'  3.66  grams  if  absolutely  pure. 

*  Longer  heating  is  to  be  avoided. 


36  PHYSIOLOGICAL    CHEMISTRY 

at  room  temperature,  crystallization  begins  and  is  complete  in  two  hours.  By 
scratching  the  sides  of  the  flask  or  by  inoculation,  the  solution  quickly  congeals  to 
form  a  thick  paste  of  reddish-yellow  silky  needles.  These  are  the  crystals  of  methyl- 
phenyljructosazone.  They  may  be  recrystallized  from  hot  95  per  cent  alcohol  and 
melt  at  i53°C. 

CHoOH 

GALACTOSE,  (CH0H)4 

! 

CHO 

Galactose  occurs  with  glucose  as  one  of  the  products  of  the  hydro- 
lysis of  lactose.  It  is  dextro-rotatory,  forms  an  osazone  with  phenyl- 
hydrazine  and  ferments  slowly  with  yeast.  Upon  oxidation  with  nitric 
acid  galactose  yields  mucic  acid,  thus  differentiating  this  monosac- 
charide from  glucose  and  fructose.  Lactose  also  yields  mucic  acid 
under  these  conditions.  The  mucic  acid  test  may  be  used  in  urine 
examination  to  differentiate  lactose  and  galactose  from  other  reducing 
sugars. 

Experiments  on  Galactose 

1 .  Phloroglucinol-Hydrochloric  Acid  Reaction  (Tollens) . — To  equal  volumes  of 
galactose  solution  and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol, 
and  heat  the  mixture  on  a  boiling  water-bath.  Galactose,  pentose  and  glycuronic 
acid  will  be  indicated  by  the  appearance  of  a  red  color.  Galactose  may  be 
differentiated  from  the  two  latter  substances  in  that  its  solutions  exhibit  no 
absorption  bands  upon  spectroscopical  examination. 

2.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing  galactose  with 
20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and  evaporate  the  mixture  in  a  broad, 
shallow  glass  vessel  on  a  boiUng  water-bath  until  the  volume  of  the  mixture  has 
been  reduced  to  about  20  c.c.  At  this  point  the  fluid  should  be  clear,  and  a  fine 
white  precipitate  of  mucic  acid  should  form. 

If  the  percentage  of  galactose  present  is  low  it  may  be  necessary  to 
cool  the  solution  and  permit  it  to  stand  for  some  time  before  the 
precipitate  will  form.  It  is  impossible  to  differentiate  between  galactose 
and  lactose  by  this  test,  but  the  reaction  serves  to  differentiate  these 
two  sugars  from  all  other  reducing  sugars.  Differentiate  lactose  from 
galactose  by  means  of  Barfoed's  test  (page  30). 

3.  Phenylhydrazine  Reaction. — Make  the  test  according  to  directions  given 
under  Glucose,  3  or  4,  pages  22  and  23. 

Pentoses,  CsHioOs 

In  plants,  and  more  particularly  in  certain  gums,  very  complex  car- 
bohydrates, called  pentosans  (see  page  50),  occur.     These  pentosans 


CARBOHYDRATES  37 

through  hydrolysis  by  acids  may  be  transformed  into  pentoses.  Pento- 
ses do  not  ordinarily  occur  in  the  animal  organism,  but  have  been  found 
in  the  urine  of  morphine  habitues  and  others,  their  occurrence  some- 
times being  a  persistent  condition  without  known  cause.  They  may  be 
obtained  from  the  hydrolysis  of  nucleoproteins  being  present  in  the 
nucleic  acid  complex  of  the  molecule.  Pentoses  are  non-fermentable, 
have  strong  reducing  power  and  form  osazones  with  phenylhydrazine. 
Pentoses  are  an  important  constituent  of  the  dietary  of  herbivorous 
animals.  Glycogen  is  said  to  be  formed  after  the  ingestion  of  these 
sugars  containing  five  oxygen  atoms.  This,  however,  has  not  been 
conclusively  proven.  On  distillation  with  strong  hydrochloric  acid 
pentoses  and  pentosans  yield  furfurol,  which  can  be  detected  by  its 
characteristic  red  reaction  with  aniline-acetate  paper. 

CHoOH 

ARABINOSE,    rCH0H)3 

I 

CHO 

Arabinose  is  one  of  the  most  important  of  the  pentoses.  The 
^arabinose  may  be  obtained  from  gum  arable,  plum  or  cherry  gum  by 
boiling  for  10  minutes  with  concentrated  hydrochloric  acid.  This 
pentose  is  dextro-rotatory,  forms  an  osazone  and  has  reducing  power, 
but  does  not  ferment.  The  i-arabinose  has  been  isolated  from  the 
urine  and  yields  an  osazone  which  melts  at  i66°-i68°C. 

Experiments  on  Arabinose 

I.  Orcinol-Hydrochloric  Acid  Reaction  (Bial).' — To  5  c.c.  of  Bial's  reagent-  in 
a  test-tube  add  2-3  c.c.  of  the  arabinose  solution  and  heat  the  mixture  gently  until 
the  first  bubbles  rise  to  the  surface.  Immediately  or  upon  cooUng  the  solution 
becomes  green  and  a  fiocculent  precipitate  of  the  same  color  may  form.  (For 
further  discussion  see  Chapter  XXIII.)  The  test  may  also  be  performed  by 
adding  the  pentose  to  the  hot  reagent. 

It  is  claimed  that  this  test  is  more  delicate  than  the  original  orcinol 
test  (see  3)  and  more  accurate,  since  menthol,  kreosotal,  etc.,  respond 
to  the  original  orcinol  test  but  not  to  Bial's.  Sachs^  has  offered 
suggestions  as  to  modification  of  the  test  in  order  to  avoid  confusion 
with  glycuronic  acid. 

iBial:  Dent.  mcd.  Woch.,  28,  252,  1902,  and  Bcrl.  kliii.  U'lnli.,  No.  18,  1903. 
-  Orcinol 1.5  gram. 

Fuming  HCl 500  grams. 

Ferric  chloride  (10  per  cent)  20-30  drops. 
^  Sachs:  Block.  Zc'U.,  i,  383,  1906,  and  2,  245,  1906. 


38  PHYSIOLOGICAL   CHEMISTRY 

2.  Phloroglucinol-Hydrochloric  Acid  Reaction  (Tollens). — To  equal  volumes  of 
arabinose  solution  and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol 
and  heat  the  mixture  on  a  boiling  water-bath.  Galactose,  pentose  or  glycuronic 
acid  will  be  indicated  by  the  appearance  of  a  red  color.  To  differentiate  between 
these  bodies  make  a  spectroscopic  examination  and  look  for  the  absorption  band 
between  D  and  E  given  by  pentoses  and  glycuronic  acid.  Differentiate  between 
the  two  latter  bodies  by  the  melting-points  of  their  osazones. 

Compare  the  reaction  with  that  obtained  with  galactose  (page  36). 

3.  Orcinol  Test. — Repeat  2,  using  orcinol  instead  of  phloroglucinol.  A  suc- 
cession of  colors  from  red  through  reddish  blue  to  green  is  produced.  A  green  pre- 
cipitate is  formed  which  is  soluble  in  amyl  alcohol  and  has  absorption  bands  be- 
tween C  and  D. 

4.  Phenylhydrazine  Reaction. — Make  this  test  on  the  arabinose  solution 
according  to  directions  given  under  Glucose,  3  or  4,  pages  22  and  23. 

CH2OH 
XYLOSE,  (CH0H)3 

CHO 

Xylose,  or  wood  sugar,  is  obtained  by  boiling  wood  gums  with  dilute 
acids  as  explained  under  Arabinose,  page  37.  It  is  dextro-rotatory, 
forms  an  osazone  and  has  reducing  power,  but  does  not  ferment. 

Experiments  on  Xylose 
1-4.  Same  as  for  arabinose  (see  above). 

RHAMNOSE,  C6H12O5 

Rhamnose  or  methyl-pentose  is  an  example  of  a  true  carbohydrate 
which  does  not  have  the  H  and  0  atoms  present  in  the  proportion  to 
form  water.  Its  formula  is  C6H12O5.  It  has  been  found  that  rham- 
nose when  ingested  by  rabbits  or  hens  has  a  positive  influence  upon  the 
formation  of  glycogen  in  those  organisms. 

DISACCHAKCDES,  C12H22O11 

The  disaccharides  as  a  class  may  be  divided  into  two  rather  dis- 
tinct groups.  The  first  group  would  include  those  disaccharides  which 
are  found  in  nature  as  such,  e.g.,  sucrose  and  lactose,  and  the  second 
group  would  include  those  disaccharides  formed  in  the  hydrolysis  of 
more  complex  carbohydrates,  e.g.,  maltose  and  iso-maltose. 

The  disaccharides  have  the  general  formula  C12H22O11,  to  which, 
in  the  process  of  hydrolysis,  a  molecule  of  water  is  added  causing  the 


CARBOHYDRATES  39 

single  disaccharide  molecule  to  split  into  two  monosaccharide  (hexose) 
molecules.  The  products  of  the  hydrolysis  of  the  more  common  di- 
saccharides  are  as  follows: 

Maltose  =  glucose+glucose. 
Lactose  =  glucose+galactose. 
Sucrose  =  glucose+f  ructose. 

All  of  the  more  common  disaccharides  except  sucrose  have  the  power 
of  reducing  certain  metallic  oxides  in  alkaline  solution,  notably  those 
of  copper  and  bismuth.  This  reducing  power  is  due  to  the  presence 
of  the  aldehyde  group  (^ — CHO)  in  the  sugar  molecule. 

MALTOSE,  Ci2H220n 

Maltose  or  malt  sugar  is  formed  in  the  hydrolysis  of  starch  through 
the  action  of  an  enzyme,  vegetable  amylase  {diastase),  contained  in  sprout- 
ing barley  or  malt.  Certain  enzymes  in  the  saliva  and  in  the  pancreatic 
juice  may  also  cause  a  similar  hydrolysis.  Maltose  is  also  an  intermedi- 
ate product  of  the  action  of  dilute  mineral  acids  upon  starch.  It  is 
strongly  dextro-rotatory,  reduces  metallic  oxides  in  alkaline  solution 
and  is  fermentable  by  yeast  after  being  inverted  (see  Polysaccharides, 
page  43)  by  the  enzyme  maltase  of  the  yeast.  In  common  wdth  the  other 
disaccharides,  maltose  may  be  hydrolyzed  with  the  formation  of  two 
molecules  of  monosaccharide.  In  this  instance  the  products  are  two 
molecules  of  glucose.  With  phenylhydrazine  maltose  forms  an  osa- 
zone,  maltosazone.  The  following  formula  represents  the  probable 
structure  of  maltose: 

CH2OH  CHO 

CHOH  CHOH 

I  ! 

CHO  CHOH 

I  I 

CHOH  CHOH 

CHOH  CHOH 

I     ,  I 

C  C CHo 

\ 


H 


Maltose. 


Experiments  on  Maltose 

1-6.  Repeat  Solubility,  Fehling's,   Nylander's,   Phenylhydrazine,  Barfoed's 
and  Fermentation  tests  as  given  under  Glucose,  pages  21-31. 


40  PHYSIOLOGICAL    CHEMISTRY 

ISO-MALTOSE,  C12H22O11 

Iso-maltose,  an  isomeric  form  of  maltose,  is  formed,  along  with  mal- 
tose by  the  action  of  diastase  upon  starch  paste,  and  also  by  the  action 
of  hydrochloric  acid  upon  glucose.  It  also  occurs  with  maltose  as  one 
of  the  products  of  salivary  digestion.  It  is  dextro-rotatory  and  with 
phenylhydrazine  gives  an  osazone  which  is  characteristic.  Iso-maltose 
is  very  soluble  and  reduces  the  oxides  of  bismuth  and  copper  in  alkaline 
solution.     Pure  iso-maltose  is  probably  only  slightly  fermentable. 

LACTOSE,  C12H22O11 

Lactose  or  milk  sugar  occurs  ordinarily  only  in  milk,  but  has  often 
been  found  in  the  urine  of  women  during  pregnancy  and  lactation.  It 
may  also  occur  in  the  urine  of  normal  persons  after  the  ingestion  of 
unusually  large  amounts  of  lactose  in  the  food.  It  has  a  strong  reducing 
power,  is  dextro-rotatory  and  forms  an  osazone  with  phenylhydrazine. 
Upon  hydrolysis  lactose  yields  one  molecule  of  glucose  and  one  molecule 
of  galactose. 

In  the  souring  of  milk  the  bacterium  lactis  and  certain  other  micro- 
organisms bring  about  lactic  acid  fermentation  by  transforming  the  lac- 
tose of  the  milk  into  lactic  acid, 

H    OH 
H— C— C— COOH, 

H    H 

and  alcohol.  This  same  reaction  may  occur  in  the  alimentary  canal  as 
the  result  of  the  action  of  putrefactive  bacteria.  In  the  preparation 
of  kephyr  and  koumyss  the  lactose  of  the  milk  undergoes  alcoholic 
fermentation,  through  the  action  of  ferments  other  than  yeast,  and  at 
the  same  time  lactic  acid  is  produced.  Lactose  and  galactose  yield 
mucic  acid  on  oxidation  with  nitric  acid.  This  fact  is  made  use  of  in 
urine  analysis  to  facihtate  the  differentiation  of  these  sugars  from  other 
reducing  sugars. 

Lactose  is  not  fermentable  by  pure  yeast. 

Experiments  on  Lactose 

1-6.  Repeat  Solubility,  Fehling's,  Phenylhydrazine,  Barfoed's,  Nylander's 
and  Fermentation  tests  as  given  under  Glucose,  pages  21-31. 

7.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  solution  containing  lactose  with 
20  c.c.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  and  evaporate  the  mixture  in  a 
broad,  shallow  glass  vessel  on  a  boiUng  water-bath,  until  the  volume  of  the  mix- 
ture has  been  reduced  to  about  20  c.c.  At  this  point  the  fluid  should  be  clear, 
and  a  fine  white  precipitate  of  mucic  acid  should  form. 


CARBOHYDRATES  4 1 

If  the  percentage  of  lactose  present  is  low  it  may  be  necessary  to 
cool  the  solution  and  permit  it  to  stand  for  some  time  before  the 
precipitate  will  appear.  It  is  impossible  to  differentiate  between  lactose 
and  galactose  by  this  test,  but  the  reaction  serves  to  differentiate  these 
two  sugars  from  all  other  reducing  sugars. 

Differentiate  lactose  from  galactose  by  means  of  Barfoed's  test, 
page  30. 

SUCROSE,  C12H22O11 

Sucrose,  also  called  saccharose  or  cane  sugar,  is  one  of  the  most 
important  of  the  sugars  and  occurs  very  extensively  distributed  in 
plants,  particularly  in  the  sugar  cane,  sugar  beet,  sugar  millet  and  in 
certain  palms  and  maples. 

Sucrose  is  dextro-rotatory  and  upon  hydrolysis,  as  before  mentioned, 
the  molecule  of  sucrose  takes  on  a  molecule  of  water  and  breaks  down 
into  two  molecules  of  monosaccharide.  The  monosaccharides  formed  in 
this  instance  are  glucose  and  fructose.     This  is  the  reaction: 

C12H22O11-I-H2O — >C6Hi206-l-C6Hi206. 

Sucrose  Glucose  Fructose 

This  process  is  called  inversion  and  may  be  produced  by  bacteria,  en- 
zymes, and  certain  weak  acids.  After  this  inversion  the  previously 
strongly  dextro-rotatory  solution  becomes  levo-rotatory.  This  is  due 
to  the  fact  that  the  fructose  molecule  is  more  strongly  levo-rotatory 
than  the  glucose  molecule  is  dextro-rotatory.  The  product  of  this 
inversion  is  called  iiivert  sugar. 

Sucrose  does  not  reduce  metalHc  oxides  in  alkahne  solution  and  forms 
no  osazone  with  phenylhydrazine.  It  is  not  fermentable  directly  by 
yeast,  but  must  first  be  inverted  by  the  enzyme  sucrose  {invertase  or 
invertin)  contained  in  the  yeast.  The  probable  structure  of  sucrose 
may  be  represented  by  the  following  formula.  Note  the  absence  of 
any  free  ketone  or  aldehyde  group. 

CH2OH  CH2OH 

I  I 

CHOH  CHO 

CHO  CHOH 

CHOH  CHOH 

I  I 

CHOH  C 

C  '  O        CH2OH 

\ 
\ 

H 

Sucrose. 


42 


PHYSIOLOGICAL   CHEMISTRY 


Experiments  on  Sucrose 

1-6.  Repeat  Solubility,  Fehling's,  Nylander's,  Barfoed's,  Phenylhydrazine 
and  Fermentation  tests  according  to  the  directions  given  under  Glucose,  pages 
21-31. 

7.  Inversion  of  Sucrose. — To  25  c.c.  of  sucrose  solution  in  a  beaker  add  5 
drops  of  concentrated  H2SO4  and  boil  one  minute-  Cool  the  solution  and  render 
neutral  with  saturated  barium  hydroxide.  Filter  off  the  precipitate  of  baiiimi 
sulphate  and  upon  the  resulting  fluid  repeat  the  phenylhydrazine,  Fehling, 
Nylander's  and  Barfoed's  reactions  as  given  under  Glucose,  pp.  26,  29  and  30 ; 
the  Resorcinol-Hydrochloric  Acid  Reaction  (Seliwanoff),  as  given  under  Fruc- 
tose, page  35.    Explain  the  results. 

8.  Alcoholic  Fermentation. — Prepare  500  c.c.  of  a  concentrated  (10-20  per 
cent)  solution  of  sucrose,  add  a  small  amount  of  egg  albumin  or  commercial 

peptone  and  introduce  the  mixture  into  a  liter  flask. 
Add  yeast,  and  by  means  of  a  bent  tube  connect  this 
flask  with  a  second  flask  containing  a  solution  of 
barium  hydroxide  protected  from  the  air  by  a  soda- 
lime  tube  in  the  stopper.  Place  the  flasks  in  a 
warm  place  and  note  the  passage  of  gas  bubbles 
into  the  barium  hydroxide  solution.  As  these  gas 
bubbles  (CO 2)  enter,  a  white  precipitate  of  barium 
carbonate  will  form.  The  sucrase  has  been  changed 
to  glucose  and  fructose  and  these  sugars  have  been 
fermented  according  to  the  following  equation : 

Fig.  8.-I0DOFORM.  {Aulenrieth.)  C6Hi20^^2C2H60H+C02 

When  the  activity  of  yeast  has  practically  ceased,  connect  the  fermentation 
flask  with  a  condenser  and  distil  the  mixture.  Catch  the  first  portion  of  the  dis- 
tillate separately  and  test  for  alcohol  by  one  of  the  following  reactions : 

(a)  Iodoform  Test. — Render  2-3  c.c.  of  the  distillate  alkaline  wtih  potassivun 
hydroxide  solution  and  add  a  few  drops  of  iodine  solution.  Heat  gently  and  note 
the  formation  of  iodoform  crystals.  Examine  these  crystals  under  the  microscope 
and  compare  them  with  those  in  Fig.  8. 

(b)  Aldehyde  Test. — Place  5  c.c.  of  the  distillate  in  a  test-tube,  add  a  few 
drops  of  potassium  dichromate  solution,  K2Cr207,  and  render  it  acid  with  dilute 
sulphuric  acid.  Boil  the  acid  solution  and  note  the  odor  of  aldehyde  changing 
to  that  of  acetic  acid. 


TRISACCHARIDES,  C18H32O16 

RAFFINOSE 

This  trisaccharide,  also  called  meKtose,  or  melitriose  occurs  in  cotton 
seed,  Australian  manna,  and  in  the  molasses  from  the  preparation  of 
beet  sugar.  It  is  dextro-rotatory,  does  not  reduce  Fehling's  solution, 
and  is  only  partly  fermentable  by  yeast. 

Raflfinose  may  be  hydrolyzed  by  weak  acids  the  same  as  the  poly- 


CARBOHYDRATES  .  43 

saccharides  are  hydrolyzed,  the  products  being  fructose  and  mehbiose; 
further  hydrolysis  of  the  mehbiose  yields  glucose  and  galactose. 

POLYSACCHARIDES,  (CeHioOg), 

In  general  the  polysaccharides  are  amorphous  bodies,  a  few,  how- 
ever, are  crystallizable.  Through  the  action  of  certain  enzjnnes  or 
weak  acids  the  polysaccharides  may  be  hydrolyzed  with  the  formation 
of  monosaccharides.  As  a  class  the  polysaccharides  are  quite  insoluble 
and  are  non-fermentable  until  inverted.  By  inversion  is  meant  the 
hydrolysis  of  disaccharide  or  polysaccharide  sugars  to  form  monosacchar- 
ides, as  indicated  in  the  following  equations: 

(a)  Ci2H220ii  +  H20-^2(G6Hi206). 

(b)  CeHioOs+H.O-^CeHisOe. 

STARCH,  (CeHioOis)^ 

Starch  is  widely  distributed  throughout  the  vegetable  kingdom, 
occuriing  in  grains,  fruits,  and  tubers.  It  occurs  in  granular  form,  the 
microscopical  appearance  being  t}^ical  for  each  individual  starch. 
The  granules,  which  differ  in  size  according  to  the  source,  are  composed 
of  alternating  concentric  rings  of  granulose  and  cellulose.  Ordinary 
starch  is  insoluble  in  cold  water,  but  if  boiled  with  water  the  cell  walls 
are  ruptured  and  starch  paste  results.  In  general  starch  gives  a  blue 
color  with  iodine. 

Starch  is  acted  upon  by  amylases,  e.g.,  salivary  amylase  (ptyalin) 
and  pancreatic  amylase  (amylopsin),  with  the  formation  of  soluble 
starch,  erythro-dextrin,  achroo-dextrins  and  maltose  (see  Salivary  Diges- 
tion, page  56).  Maltose  is  the  principal  end-product  of  this  enzyme 
action.  Upon  boiling  a  starch  solution  with  a  dilute  mineral  acid  a 
series  of  similar  bodies  is  formed,  but  under  these  conditions  glucose 
is  the  principal  end-product. 

Soluble  starch  may  be  prepared  by  the  action  of  dilute  hydro- 
chloric acid  on  ordinary  starch  for  several  weeks,  or  at  higher  tem- 
peratures for  a  shorter  period.  By  precipitation  with  alcohol  this  may 
be  obtained  in  a  dry  form  readily  soluble  in  cold  water.^ 

Experiments  on  Starch 

I.  Preparation  of  Potato  Starch. — ^Pare  a  raw  potato,  comminute  it  upon  a  fine 
grater,  mix  with  water,  and  "whip  up"  the  pulped  material  vigorously  before 
straining  it  through  cheese  cloth  or  gauze  to  remove  the  coarse  particles.     The 

^  Fernbach:  Proceedings  St/i  Int.  Cong.  Appl.  Chcin.,  13,  131,  1912. 
Chapin:  Jour.  Ind.  and  Eng.  Chem.,  6,  649,  1914. 


44 


PHYSIOLOGICAL    CHEMISTRY 


Fig.  9. — Potato. 


Fig.  10. — Bean.  Fig.  ii. — Arrowroot. 


Fig.  12. — Rye. 


Fig.  13. — Barley. 


Fig.  14. — Oat. 


Fig.  15. — Buckwheat.  Fig.  16.^ — ^Maize. 


Fig.  17. — Rice. 


Fig.  18.— Pea. 


Fig.  19. — Wheat. 


Starch  Granules  from  Various  Sources.     {Lcffmann  and  Beam.) 


CARBOHYDRATES  45 

starch  rapidly  settles  to  the  bottom  and  can  be  washed  by  repeated  decantation. 
Allow  the  compact  mass  of  starch  to  drain  thoroughly  and  spread  it  out  on  a  watch 
glass  to  dry  in  the  air.  If  so  desired  this  preparation  may  be  used  in  the  experi- 
ments which  follow. 

2.  Microscopical  Examination.  Examine  microscopically  the  granules  of  the 
various  starches  submitted  and  compare  them  with  those  shown  in  Figs.  9-19, 
page  44.  The  suspension  of  the  granules  in  a  drop  of  water  will  faciUtate  the 
microscopical  examination. 

3.  Solubility. — Try  the  solubiUty  of  one  form  of  starch  in  each  of  the  ordinary 
solvents  (see  page  21).  If  uncertain  regarding  the  solubility  in  any  reagent, 
filter  and  test  the  filtrate  with  iodine  solution  as  given  under  5  below.  The  pro- 
duction of  a  blue  color  would  indicate  that  the  starch  had  been  dissolved  by  the 
solvent.  J 

4.  Iodine  Test. — Place  a  few  granules  of  starch  in  one  of  the  depressions  of  a 
porcelain  test-tablet  and  treat  with  a  drop  of  a  dilute  solution  of  iodine  in  potas- 
sium iodide.  The  granules  are  colored  blue  due  to  the  formation  of  so-called 
iodide  of  starch.  The  cellulose  of  the  granule  is  not  stained  as  may  be  seen  by 
examining  microscopically. 

5.  Iodine  Test  on  Starch  Paste.  ^ — Repeat  the  iodine  test  using  the  starch 
paste.  Place  2-3  c.c.  of  starch  paste-  in  a  test-tube,  add  a  drop  of  the  dilute 
iodine  solution  and  observe  the  production  of  a  blue  color.  Heat  the  tube  and 
note  the  disappearance  of  the  color.     It  reappears  on  cooling. 

In  similar  tests  note  the  influence  of  alcohol  and  of  alkali  upon  the  so-called 
iodide  of  starch. 

The  composition  of  the  iodide  of  starch  is  not  definitely  known.  In  per- 
forming this  test  the  solution  must  always  be  neutral  or  acid  in  reaction. 

6.  Fehling's  Test. — On  starch  paste  (see  page  26). 

7.  Hydrolysis  of  Starch. — Place  about  25  c.c.  of  starch  paste  in  a  small 
beaker,  add  10  drops  of  concentrated  HCl,  and  boil.  By  means  of  a  small  pipette, 
at  the  end  of  each  minute',  remove  a  drop  of  the  solution  to  the  test-tablet  and 
make  the  regular  iodine  test.  As  the  testing  proceeds  the  blue  color  should 
gradually  fade  and  finally  disappear.  At  this  point,  after  cooUng  and  neutraUz- 
ing  with  soUd  KOH,  FehUng's  test  (see  page  26)  should  give  a  positive  result 
due  to  the  formation  of  a  reducing  sugar  from  the  starch.  Make  the  phenyl- 
hydrazine  test  upon  some  of  the  hydrolyzed  starch.  What  sugar  has  been 
formed? 

8.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid  solution  to  a  small 
amount  of  starch  paste  in  a  test-tube.  The  liquid  will  become  strongly  opaque 
and  ordinarily  a  yellowish-white  precipitate  is  produced.  Compare  this  result  with 
the  result  of  the  similar  experiment  on  dextrin  (page  48). 

9.  Diffusibility  of  Starch  Paste. — -Test  the  diffusibility  of  starch  paste  through 
animal  memljrane,  parchment  paper  or  collodion,  making  a  dialyzer  like  one  of  the 
models  shown  in  Fig.  2,  page  24. 

^  Preparation  of  Starch  Paste. — Grind  2  grams  of  starch  powder  in  a  mortar  with  a  small 
amount  of  cold  water.  Bring  200  c.c.  of  water  to  the  boiling-point  and  add  the  starch  m\x- 
ture  from  the  mortar  with  continuous  stirring.  Bring  again  to  the  boiling-point  and  allow 
it  to  cool.  This  makes  an  approximate  i  per  cent  starch  paste  which  is  a  very  satisfactory 
strength  for  general  use. 

-  For  this  particular  test  a  starch  paste  of  very  satisfactory  strength  may  be  made  by 
mixing  i  c.c.  of  a  i  per  cent  starch  paste  with  100  c.c.  of  water. 


46  PHYSIOLOGICAL   CHEMISTRY 

INULm,  (C6Hio05)x 

Inulin  is  a  polysaccharide  which  may  be  obtained  as  a  white,  odor- 
less, tasteless  powder  from  the  tubers  of  the  artichoke,  elecampane,  or 
dahlia.  It  has  also  been  prepared  from  the  roots  of  chicory,  dandelion, 
and  burdock.  It  is  very  slightly  soluble  in  cold  water  and  quite  easily 
soluble  in  hot  water.  In  cold  alcohol  of  60  per  cent  or  over  it  is  prac- 
tically insoluble.  Inulin  gives  a  negative  reaction  with  iodine  solution. 
The  "yellow"  color  reaction  with  iodine  mentioned  in  many  books  is 
doubtless  merely  the  normal  color  of  the  iodine  solution.  It  is  very 
difl&cult  to  prepare  inulin  which  does  not  reduce  Fehling's  solution 
slightly.  This  reducing  power  may  be  due  to  an  impurity.  Prac- 
tically all  commercial  preparations  of  inulin  possess  considerable 
reducing  power. 

Inulin  is  levo-rotatory  and  upon  hydrolysis  by  acids  or  by  the 
enzyme  inulase  it  yields  the  monosaccharide  fructose  which  readily 
reduces  Fehling's  solution.  The  ordinary  amylolytic  enzymes  occur- 
ring in  the  animal  body  do  not  digest  inulin.  A  small  part  of  the 
ingested  inulin  may  be  hydrolyzed  by  the  acid  gastric  juice,  but  Lewis^ 
has  recently  shown  that  "the  value  of  inulin  as  a  significant  source  of 
energy  in  human  dietaries  must  be  questioned." 

Experiments  on  Inulin 

1.  Solubility. — Try  the  solubility  of  inulin  powder  in  hot  and  cold  water  and 
alcohol.  If  uncertain  regarding  the  solubiUty  in  any  reagent,  filter  and  neutralize 
the  filtrate  if  it  is  alkaline  in  reaction.  Add  a  drop  of  concentrated  hydrochloric 
acid  to  the  filtrate  and  boil  it  for  one  minute.  Render  the  solution  neutral  or 
slightly  alkaline  with  solid  potassium  hydroxide  and  try  Fehling's  test.  What 
is  the  significance  of  a  positive  Fehling's  test  in  this  connection? 

2.  Iodine  Test. — (a)  Place  2-3  c.c.  of  the  inuHn  solution  in  a  test-tube  and 
add  a  drop  of  dilute  iodine  solution.    What  do  you  observe  ? 

(b)  Place  a  small  amount  of  inulin  powder  in  one  of  the  depressions  of  a  test- 
tablet  and  add  a  drop  of  dilute  iodine  solution.  Is  the  effect  any  different  from 
that  observed  above? 

3.  MoUsch's  Reaction. — Repeat  this  test  according  to  directions  given  under 
Glucose,   2,  page   21. 

4.  Fehhng's  Test, — Make  this  test  on  the  inuUn  solution  according  to  the 
instructions  given  under  Glucose,  page  26.     Is  there  any  reduction? ^ 

5.  Hydrolysis  of  Inulin. — Place  5  c.c.  of  inulin  solution  in  a  test-tube,  add  a 
drop  of  concentrated  hydrochloric  acid  and  boil  it  for  one  minute.  Now  cool 
the  solution,  neutralize  it  with  concentrated  KOH  and  test  the  reducing  action 
of  I  c.c.  of  the  solution  upon  i  c.c.  of  diluted  (1:4)  Fehhng's  solution.    Also 

^  Lewis:  Journal  American  Medical  Ass'n.,  58,  1176,  1912. 
^  See  the  discussion  of  the  properties  of  inulin,  above. 


CARBOHYDRATES  47 

try  the  Resorcinol-Hydrochloric  Acid  reaction  as  given  on  p.  35.     Explain  the 
result.' 

GLYCOGEN,    (CeHioO^)^ 

(For  discussion  and  experiments  see  Muscular  Tissue,  Chapter  XIX.) 

LICHENIN,   (CeHioOs) 

Lichenin  may  be  obtained  from  Cetraria  islandica  (Iceland  moss). 
It  forms  a  difficultly  soluble  jelly  in  cold  water  and  an  opalescent  solu- 
tion in  hot  water.  It  is  optically  inactive  and  gives  no  color  with 
iodine.  Upon  hydrolysis  with  dilute  mineral  acids  lichenin  yields  dex- 
trins  and  glucose.  It  is  said  to  be  most  nearly  related  chemically  to 
starch.  Saliva,  pancreatic  juice,  malt  diastase  and  gastric  juice  have 
no  noticeable  action  on  lichenin. 

DEXTRIN,    (CeHioOs)^ 

The  dextrins  are  the  bodies  formed  midway  in  the  stages  of  the 
hydrolysis  of  starch  by  weak  acids  or  an  enzyme.  They  are  amorphous 
bodies  which  are  easily  soluble  in  water,  acids,  and  alkalis,  but  are  in- 
soluble in  alcohol  or  ether.  Dextrins  are  dextro-rotatory  and  are  not 
fermentable  by  yeast. 

The  dextrins  may  be  hydrolyzed  by  dilute  acids  to  form  glucose 
and  by  amylases  to  form  maltose.  With  iodine  one  form  of  dextrin 
(erythro-dextrin)  gives  a  red  color.  Their  power  to  reduce  Fehling's 
solution  is  questioned.  The  lower  members  of  the  dextrin  series  prob- 
ably reduce. 

Experiments  on  Dextrin 

1.  SolubiHty. — Test  the  solubiUty  of  pulverized  dextrin  in  hot  and  cold  water. 
Dextrin  forms  a  clear  solution  in  hot  water,  distinguishing  it  from  glycogen  which 
gives  an  opalescent  solution. 

2.  Iodine  Test. — Place  a  drop  of  dextrin  solution  in  one  of  the  depressions 
of  the  test-tablet  and  add  a  dilute  solution  of  iodine  in  potassiimi  iodide.  A  red 
color  results  due  to  the  formation  of  the  red  iodide  of  dextrin.  Ordinary  dextrin 
preparations  contain  some  starch  and  in  the  presence  of  starch  it  is  necessary  to 
have  an  excess  of  iodine  present.  If  the  reaction  is  not  sufficiently  pronounced 
make  a  stronger  solution  from  pulverized  dextrin  and  repeat  the  test.  The 
solution  should  be  slightly  acid  to  secure  the  best  results. 

Make  proper  tests  to  show  that  the  red  iodide  of  dextrin  is  influenced  by 

^  If  the  inulin  solution  gave  a  positive  Fehling  test  in  the  last  experiment  it  will  be  neces- 
sary to  check  the  hydrolysis  experiment  as  follows:  To  5  c.c.  of  the  inulin  solution  in  a  test- 
tube  add  one  drop  of  concentrated  hj'drochloric  acid,  neutralize  with  concentrated  KOH 
solution  and  test  the  reducing  action  of  i  c.c.  of  the  resulting  solution  upon  i  c.c.  of  diluted 
(i  :4)  Fehling's  solution.  This  will  show  the  normal  reducing  power  of  the  inulin  solution. 
In  case  the  inulin  was  hydrolyzed,  the  Fehling's  test  in  the  hydrolysis  experiment  should 
show  a  more  pronounced  reduction  than  that  observed  in  the  check  experiment. 


48  PHYSIOLOGICAL   CHEMISTRY 

heat,  alkali,  and  alcohol  in  a  similar  manner  to  the  blue  iodide  of  starch  (see 
page  45). 

The  color  in  the  case  of  dextrin  does  not  reappear  as  readily  on  cooling  as 
in  the  case  of  starch. 

3.  Fehhng's  Test. — See  if  the  dextrin  solution  will  reduce  Fehling's  solution. 

4.  Hydrolysis  of  Dextrin. — Take  25  c.c.  of  dextrin  solution  in  a  small  beaker, 
add  5  drops  of  dilute  hydrochloric  acid,  and  boil.  By  means  of  a  small  pipette, 
at  the  end  of  each  minute,  remove  a  drop  of  the  solution  to  one  of  the  depressions 
of  the  test-tablet  and  make  the  iodine  test.  The  power  of  the  solution  to  produce 
a  color  with  iodine  should  rapidly  disappear.  When  a  negative  reaction  is  ob- 
tained cool  the  solution  and  neutraUze  it  with  concentrated  potassium  hydroxide. 
Try  Fehhng's  test  (see  page  26).  This  reaction  is  now  strongly  positive,  due  to 
the  formation  of  a  reducing  sugar.  Determine  the  nature  of  the  sugar  by  means 
of  the  phenylhydrazine  test  (see  pages  22  and  23). 

5.  Precipitation  by  Alcohol. — To  about  50  c.c.  of  95  per  cent  alcohol  in  a  small 
beaker  add  about  10  c.c.  of  a  concentrated  dextrin  solution.  Dextrin  is  thrown 
out  of  solution  as  a  gummy  white  precipitate.  Compare  the  result  with  that 
obtained  under  Glucose,  7,  page  24. 

6.  Influence  of  Tannic  Acid. — Add  an  excess  of  tannic  acid  solution  to  a 
small  amount  of  dextrin  solution  in  a  test-tube.  No  precipitate  forms.  This 
result  differs  from  the  result  of  the  similar  experiment  upon  starch  (see  Starch,  8, 

page  45)- 

7.  Diflfusibility  of  Dextrin. — (See  Starch,  9,  page  45.) 

CELLULOSE,  (CsHioOs)^ 

This  complex  polysarcharide  forms  a  large  portion  of  the  cell  wall 
of  plants.  It  is  extremely  insoluble  and  its  molecule  is  much  more  com- 
plex than  the  starch  molecule.  The  best  quality  of  filter  paper  and 
the  ordinary  absorbent  cotton  are  good  types  of  cellulose. 

At  one  time  there  was  but  a  single  known  solvent  for  cellulose. 
Recent  investigation  has,  however,  revealed  a  long  list  of  cellulose 
solvents.     (See  Experiment  7.) 

Cellulose  is  not  hydrolyzed  by  boiling  with  dilute  mineral  acids.  It 
may  be  hydrolyzed,  however,  by  treating  with  concentrated  sulphuric 
acid  then  subsequently  diluting  the  solution  with  water  and  boiling. 
The  product  of  this  hydrolysis  is  glucose. 

There  is  some  difference  of  opinion  as  to  the  exact  extent  to  which 
cellulose  is  utilized  in  the  animal  organism.  It  is  no  doubt,  more  effi- 
ciently utilized  by  herbivora  than  by  carnivora  or  by  man.  It  is  claimed 
that  about  25  per  cent  may  be  utilized  by  herbivora,  less  than  5  per  cent 
by  dogs  whereas  the  quantity  utilized  by  man  is  "  too  small  for  it  to  play 
a  role  of  importance  in  the  diet  of  a  normal  individual."^  In  neither 
man  nor  the  lower  animals  has  there  been  demonstrated  any  formation 

'  Swartz:  Transactions  of  the  Connecticut  Academy  of  Arts  and  Sciences,  16,  247,  1911. 


CARBOHYDRATES  49 

of  sugar  or  glycogen  from  cellulose.^  It  is  probable  that  the  cellulose 
which  disappears  from  the  intestine  is  transformed  for  the  most  part  into 
fatty  acids. - 

Experiments  on  Cellulose 

1 .  Solubility. — Test  the  solubility  of  cellulose  in  water,  dilute  and  concentrated 
acid  and  alkali. 

2.  Iodine  Test. — ^Add  a  drop  of  dilute  iodine  solution  to  a  few  shreds  of  cotton 
on  a  test-tablet.  Cellulose  differs  from  starch  and  dextrin  in  giving  no  color 
with  iodine. 

3.  Formation  of  Amyloid.'' — Add  10  c.c.  of  dilute  and  5  c.c.  of  concentrated 
H2SO4  to  some  absorbent  cotton  in  a  test-tube.  When  entirely  dissolved  fwith- 
out  heating  I  pour  one-half  of  the  solution  into  another  test-tube,  cool  it  and  dilute 
with  water.  Amyloid  forms  as  a  gxmimy  precipitate  and  gives  a  brown  or  blue 
coloration  with  iodine. 

After  allowing  the  second  portion  of  the  acid  solution  of  cotton  to  stand  about 
10  minutes,  dilute  it  with  water  in  a  small  beaker  and  boil  for  15-30  minutes. 
Now  cool,  neutraUze  with  soUd  KOH  and  test  with  Fehhng's  solution.  Glucose 
has  been  formed  from  the  cellulose  by  the  action  of  the  acid. 

4.  Ammoniacal  Cupric  Hydroxide  SolubiHty  Test  (Schweitzer). — Place  a 
little  absorbent  cotton  in  a  test-tube,  add  Schweitzer's  reagent,^  and  stir  the 
cellulose  with  a  glass  rod.  When  completely  dissolved  acidify  the  solution  with 
acetic  acid.     An  amorphous  precipitate  of  cellulose  is  produced. 

5.  Hydrochloric  Acid — Zinc  Chloride  SolubiUty  Test  (Cross  and  Bevan).^ — 
Place  a  Uttle  absorbent  cotton  in  a  test-tube,  add  Cross  and  Sevan's  reagent,^ 
and  stir  the  cellulose  with  a  glass  rod.  When  solution  is  complete  reprecipitate 
the  cellulose  with  95  per  cent  alcohol. 

6.  Iodine-Zinc  Chloride  Reaction. — Place  a  little  absorbent  cotton  or  quantita- 
tive filter  paper  in  a  test-tube  and  treat  it  with  the  iodine-zinc  chloride  reagent." 
A  blue  color  forms  on  standing.  Amyloid  has  been  formed  from  the  cellulose 
through  the  action  of  the  ZnClo  and  the  iodine  solution  has  stained  the  amyloid 
blue. 

7.  Other  Cellulose  Solvents. — It  has  recently  been  demonstrated  by  Deming' 
that  there  are  many  excellent  solvents  for  cellulose  (filter  paper).  For  example, 
the  concentrated  aqueous  solutions  of  certain  salts  such  as  antimony  trichloride, 

'Lusk:  American  Journal  of  Physiology,  27,  467,  1911;  also  Hoffmann,  Inaugural  dis- 
sertation, Halle-Wittenberg,  19 10. 

-  Tappeiner:  Zeitschrijt  fiir  Biologic,  24,  105,  1888. 

^  This  body  derives  its  name  from  amylum  (starch)  and  is  not  to  be  confounded  with 
amyloid,  the  glycoprotein. 

*  Schweitzer's  reagent  is  made  by  adding  potassium  hydroxide  to  a  solution  of , copper 
sulphate  which  contains  some  ammonium  chloride.  A  precipitate  of  cupric  hydroxide 
forms  and  this  is  filtered  off,  washed,  and  3  grams  of  the  moist  cupric  hydroxide  brought 
into  solution  in  a  liter  of  20  per  cent  ammonium  hydroxide. 

^  Cross  and  Bevan:  Chemical  Xcus,  63,  p.  06. 

*  Cross  and  Bevan's  reagent  may  be  prepared  by  combining  two  parts  of  concentrated 
hydrochloric  acid  and  one  part  of  zinc  chloride,  by  weight. 

'  The  iodine-zinc  chloride  reagent  as  suggested  by  Xowopokrowsky  (Beihefte  Botan. 
Ccnlr.,  28,  90,  191 2)  may  be  made  b)'  dissolving  20  grams  ZnCl;  in  8.5  c.c.  water  and  when 
cool  introducing  the  iodine  solution  (3  grams  KI  +  i-S  gram  I  in  Oo  c.c.  water)  drop  by  drop 
until  iodine  begins  to  precipitate. 

8  Deming:  Journal  American  Chemical  Society,  33,  1515,  191 1. 


50  PHYSIOLOGICAL   CHEMISTRY 

stannous  chloride  and  zinc  bromide.  In  hydrochloric  acid  solution  the  solvent  action 
of  the  above  salts  is  increased.  The  following  salts  are  also  good  solvents  in  hydro- 
chloric acid  solution:  mercuric  chloride,  bismuth  chloride,  antimony  pentachloride, 
tin  tetrachloride  and  titanium  tetrachloride.  In  the  case  of  the  last-mentioned  salt 
the  swollen,  transparent  character  of  the  cellulose  fibers  preliminary  to  solution 
can  be  seen  very  nicely. 

Try  selected  solvents  suggested  by  the  instructor. 

HEMICELLULOSES 

The  hemicelluloses  differ  from  cellulose  in  that  they  may  be  hydro- 
lyzed  upon  boiling  with  dilute  mineral  acids.  They  differ  from  other 
polysaccharides  in  not  being  readily  digested  by  amylases.  Hemi- 
cellulose  may  yield  pentosans,  or  hexosans  upon  hydrolysis. 

Pentosans. — Pentosans  yield  pentoses  upon  hydrolysis.  So  far  as  is 
known  they  do  not  occur  in  the  animal  kingdom.  They  have,  however, 
a  very  wide  distribution  in  the  vegetable  kingdom,  being  present 
in  leaves,  roots,  seeds  and  stems  of  all  forms  of  plants,  many  times  in 
intimate  association  or  even  chemical  combination  with  galactans.  In 
herbivora,  pentosans  are  40-80  per  cent  utilized. '^  The  few  tests  on 
record  as  to  the  pentosan  utilization  by  man^  indicate  that  80-95 
per  cent  disappear  from  the  intestine.  According  to  Cramer,^  bacteria 
are  efficient  hemicellulose  transformers.  It  has  not  yet  been  dem- 
onstrated that  pentosans  form  glycogen  in  man,  and  for  this  reason 
they  must  be  considered  as  playing  an  unimportant  part  in  human 
nutrition.  Gum  arable  an  important  pentosan  may  be  hydrolyzed 
by  concentrated  hydrochloric  acid  if  boiled  for  a  short  time.  The 
pentose  arabinose  results  from  such  hydrolysis. 

Galactans. — In  common  with  the  pentosans  the  galactans  have  a  very 
wide  distribution  in  the  vegetable  kingdom.  The  pure  galactans  yield 
galactose  upon  hydrolysis.  One  of  the  most  important  members  of 
the  galactan  group  is  agar-agar,  a  product  prepared  from  certain  types 
of  Asiatic  sea-weed.  This  galactan  is  about  50  per  cent  utilizable  by 
herbivora^  and  8-27  per  cent  utilizable  by  man.^  Agar  ingestion  has 
been  shown  to  be  a  very  efficient  therapeutic  aid  in  cases  of  chronic 
constipation.^''  This  is  particularly  true  when  the  constipation  is  due 
to  the  formation  of  dry,  hard,  fecal  masses  (scybala),  a  type  of  fecal 
formation  which  frequently  follows  the  ingestion  of  a  diet  which  is 

^  Swartz:  Transactions  of  the  Connecticut  Academy  of  Arts  and  Sciences,  16,  247,  1911. 

2  Konig  and  Reinhardt:  Zeit.f.  Untersuchung  der  Nahrwtgs  u.  Genussmittel,  5,  no,  1902. 

2  Cramer:  Inaug.  Diss.,  Halle,  1910. 

*Lohrisch:  Zeit.f.  exper.  Path.  u.  Pharm.,  5,  478,  1908. 

^  Saiki:  Jour.  Biol.  Chem.,  2,  251,  1906. 

^  Mendel:  Zentralblatf.  d.  gesammte  Phys.  u.  Path,  des  Stoffw.,  No.  17,  i,  1908. 

'Schmidt:  Miinch.  nied.  Woch.,  52,  1970,  1905. 


CARBOHYDRATES  5 1 

very  thoroughly  digested  and  absorbed.  The  agar,  because  of  its 
relative  indigestibility  and  its  property  of  absorbing  water  yields  a 
bulky  fecal  mass  which  is  sufficiently  soft  to  permit  of  easy  evacua- 
tion. Agar  has  been  used  with  good  results  in  the  treatment  of  con- 
stipation in  children.^  The  function  of  agar  is  not  limited  to  its  use 
in  connection  with  constipation,  it  may. serve  in  other  capacities  as  an 
aid  to  intestinal  therapeutics. ^ 

Experiments  on  a  Pentosan 

1.  Solubility. — Test  the  solubility  of  gum  arabic  in  hot  and  cold  water  and 
alcohol. 

2.  Iodine  Test. — ^Add  a  drop  of  dilute  iodine  solution  to  a  Uttle  gum  arabic 
on  a  test-tablet.     It  resembles  cellulose  in  giving  no  color  with  iodine. 

3.  Hydrolysis  of  Gum  Arabic. — Introduce  a  little  gum  arabic  into  a  test-tube, 
add  5-10  c.c.  of  strong  hydrochloric  acid  (cone.  HCl  and  water  1:1)  and  heat  to 
boiling  for  5-10  minutes.  Cool,  neutralize  with  potassium  hydroxide  and  test 
by  the  Fehling  or  some  other  reduction  test.  A  positive  reaction  should  be  ob- 
tained indicating  that  the  gum  arabic  has  been  hydrolyzed  by  the  acid  with  the 
production  of  a  reducing  substance.  What  is  this  reducing  substance?  How 
would  you  identify  it? 

Experiments  on  a  Galactan 

1.  SolubiUty. — Test  the  solubility  of  agar-agar  in  hot  and  cold  water.  Ob- 
serve its  marked  property  of  imbibing  water  (see  above). 

2.  Iodine  Test. — ^Add  a  drop  of  dilute  iodine  solution  to  a  little  agar-agar  on  a 
test-tablet.     It  resembles  cellulose  in  giving  no  color  with  iodine. 

3.  Hydrolysis  of  Agar-agar.^ — Introduce  a  few  pieces  of  agar-agar  into  a  test- 
tube,  add  5-10  c.c.  of  strong  hydrochloric  acid  (cone.  HCl  and  water  1:1)  and 
heat  to  boiling  for  5-10  minutes.  Cool,  neutralize  with  potassium  hydroxide  and 
test  by  the  Fehhng  or  some  other  reduction  test.  A  positive  reaction  should  be 
obtained  indicating  that  the  agar-agar  has  been  hydrolyzed  by  the  acid  with  the 
production  of  a  reducing  substance.  What  is  this  reducing  substance?  How 
would  you  identify  it? 

REVIEW  OF  CARBOHYDRATES 

In  order  to  facilitate  the  student's  review  of  tlie  carbohydrates,  the 
preparation  of  a  chart  similar  to  the  appended  model  is  recommended. 
The  signs  +  and  —  may  be  conveniently  used  to  indicate  positive 
and  negative  reaction.  Only  those  carbohydrates  which  are  of  greatest 
importance  from  the  standpoint  of  physiological  chemistry  have  been 
included  in  the  chart. 

*  Morse:  Journal  American  Medical  Ass'n.,  55,  934   1910. 
^Einhorn:  Bcrl.  klin.  Woch.,  49,  113,  1912. 


52 


PHYSIOLOGICAL   CHEMISTRY 


MODEL  CHART  FOR  REVIEW  PURPOSES 

Carbo- 
hydrate 

Solubility 

a-Naphthol  Reaction 
(Molisch) 

Moore's  Test    . 
Fehling's  Test 
Nylander-Alm6n  Test 
Barfoed's  Test 

Iodine  Test 

Resorcinol-Hydrochloric 
Acid  Reaction  (SeliwanofI) 

Orcinol-H  ydrochloric 
Acid  Reaction  (Bial) 

Mucic  Acid  Test 

Precipitation  by 
Alcohol 

a 

a 
o 

N 

0 

a 

o 

1 

DifEusiblility 

Fermentation 

Products  of  Hydrolysis 

1 

a 
Pi. 

Glucose. 

i                   i 

1 

1 

1   Fructose. 

t 

\ 

1       1 

Pentose. 

i 

! 

[ 

i 

Maltose. 

1                   1     - 

i 

'       ' 

Lactose.                                             j 

I 

j        \     ■  \        i      ^      :      1      i 

Sucrose.                                             | 

i          '          1 

'■'■          \          \ 

Starch. 

Invdin. 

1 

;       i       ; 

Glycogen. 

' 

1 

Dextrin.                                             , 

Cellulose.                      ~           | 

1          '          '          ^ 

1         ! 

Gum  Arabic. 

1 

1 

Agar-agar.                                                                                        ' 

"Unknown"  Solutions  of  Carbohydrates 

At  this  point  the  student  will  be  given  several  "unknown"  solutions, 
each  solution  containing  one  or  more  of  the  carbohydrates  studied. 
He  will  be  required  to  detect,  by  means  of  the  tests  on  the  preceding 
pages,  each  carbohydrate  constituent  of  the  several  "unknown"  solu- 
tions and  hand  in,  to  the  instructor,  a  written  report  of  his  findings,  on 
slips  furnished  by  the  laboratory. 

The  scheme  given  on  page  53  may  be  of  use  in  this  connection. 


CARBOHYDRATES 


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CHAPTER  III 
SALIVARY  DIGESTION 

The  saliva  is  secreted  by  three  pairs  of  glands,  the  submaxillary, 
sublingual,  and  parotid,  reinforced  by  numerous  small  glands  called 
buccal  glands.  The  saliva  secreted  by  each  pair  of  glands  possesses 
certain  definite  characteristics  peculiar  to  itself.  For  instance,  in  man 
the  parotid  glands  ordinarily  secrete  a  thin,  watery  fluid,  the  submaxil- 
lary glands  secrete  a  somewhat  thicker  fluid  containing  mucin,  while  the 
product  of  the  sublingual  glands  has  a  more  mucilaginous  character. 
The  saliva  as  collected  from  the  mouth  is  the  combined  product  of  all 
the  glands  mentioned.  The  fact  that  there  are  pronounced  variations 
in  the  composition  of  different  fractions  of  saliva  secreted  by  the  same 
normal  individual  on  a  uniform  diet  has  been  emphasized  by  Lothrop 
and  Gies.^ 

The  saliva  may  be  induced  to  flow  by  many  forms  of  stimuli,  such  as 
chemical,  mechanical,  electrical,  thermal,  and  psychical,  the  nature  and 
amount  of  the  secretion  depending,  to  a  limited  degree,  upon  the  par- 
ticular class  of  stimuli  employed  as  well  as  upon  the  character  of  the 
individual  stimulus.  For  example,  in  experiments  upon  dogs  it  has  been 
found  that  the  mechanical  stimulus  afforded  by  dropping  several  pebbles 
into  the  animal's  mouth  caused  the  flow  of  but  one  or  two  drops  of 
saliva,  whereas  the  mechanical  stimulus  afforded  by  sand  thrown  into 
the  mouth  induced  a  copious  flow  of  thin  watery  fluid.  Again,  when 
ice-water  or  snow  was  placed  in  the  animal's  mouth  no  saliva  was  seen, 
while  an  acid  or  anything  possessing  a  bitter  taste,  which  the  dog  wished 
to  reject,  caused  a  free  flow  of  the  thin  saliva.  On  the  other  hand,  when 
articles  of  food  were  placed  in  the  dog's  mouth  the  animal  secreted  a 
thicker  saliva  having  a  higher  mucin  content — a  fluid  which  would  lubri- 
cate the  food  and  assist  in  the  passage  of  the  bolus  through  the  esopha- 
gus. It  was  further  found  that  by  simply  drawing  the  attention  of  the 
animal  to  any  of  the  substances  named  above,  results  were  obtained 
similar  to  those  secured  when  the  substances  were  actually  placed  in  the 
animal's  mouth.  For  example,  when  a  pretense  was  made  of  throw- 
ing sand  into  the  dog's  mouth,  a  watery  saliva  was  secreted,  whereas 
food  under  the  same  conditions  excited  a  thicker  and  more  slimy 
secretion.     The  exhibition  of  dry  food,  in  which  the  dog  had  no  par- 

1  Lothrop  and  Gies:  Journal  of  I  he  Allied  {Dental)  Societies,  6,  65,  191 1. 

54 


SALIVARY    DIGESTION  55 

ticular  interest  (dry  bread),  caused  the  secretion  of  a  large  amount  of 
watery  saliva,  while  the  presentation  of  moist  food,  which  was  eagerly 
desired  by  the  animal,  called  forth  a  much  smaller  secretion,  slimy  in 
character.  These  experiments  show  it  to  be  rather  difficult  to  dif- 
ferentiate between  the  influence  of  physiological  and  psychical  stimuli. 

The  amount  of  saliva  secreted  by  an  adult  in  24  hours  has  been  vari- 
ously placed,  as  the  result  of  experiment  and  observation,  between  looo 
'and  1500  c.c,  the  exact  amount  depending,  among  other  conditions, 
upon  the  character  of  the  food. 

The  saliva  of  adults  ordinarily  has  a  weak,  alkaline  reaction  to  litmus, 
but  becomes  acid,  in  some  instances,  2-3  hours  after  a  meal  or  during 
fasting.  The  saliva  of  the  newborn  is  generally  neutral  to  litmus, 
whereas  that  of  infants,  especially  those  breast-fed,  is  generally  acid.^ 
The  alkalinity  of  saliva  is  due  principally  to  di-sodium  hydrogen  phos- 
phate (Na2HP04)  and  its  average  alkalinity  may  be  said  to  be  equiva- 
lent to  0.08-0. 1  per  cent  sodium  carbonate.  The  saliva  is  the  most  dilute 
of  all  the  digestive  secretions,  having  an  average  specific  gravity  of  1.005 
and  containing  only  0.5  per  cent  of  solid  matter.  Among  the  solids  are 
found  albumin,  globulin,  mucin,  urea,  the  enzymes  salivary  amylase 
(ptyalin),  maltase,  and  pep  tide-splitting  enzymes,  phosphates,  and 
other  inorganic  constituents.  Potassium  thiocyanate,  KSCN,  is  also 
generally  present  in  the  saliva.  It  has  been  claimed  that  this  sub- 
stance is  present  in  greatest  amount  in  the  saliva  of  habitual  smokers. 
The  significance  of  thiocyanate  in  the  saliva  is  not  known;  it  probably 
comes  from  the  ingested  thiocyanates  and  from  the  breaking  down  of 
protein  material.  The  attempts  to  show  some  relationship  between 
tooth  decay  and  the  thiocyanate  content  of  the  saliva  secreted  into 
the  mouth  cavity  have  met  with  failure.  The  most  recent  experiments- 
indicate  a  virtual  absence  of  such  relationship. 

The  so-called  tartar  formation  on  the  teeth  is  composed  almost 
entirely  of  calcium  phosphate  with  some  calcium  carbonate,  mucin, 
epithelial  cells,  and  organic  debris  derived  from  the  food.  The  calcium 
salts  are  held  in  solution  as  acid  salts,  and  are  probably  precipitated  by 
the  ammonia  of  the  breath.  The  various  organic  substances  just  men- 
tioned are  carried  down  in  the  precipitation  of  the  calcium  salts. 

The  suggestion  has  been  made  that  mucin  is  the  salivary  constituent 
*' which  is  particularly  influential  in  the  development  of  local  conditions 
favoring  the  onset  of  dental  decay.  "^ 

The  principal  enzyme  of  the  saliva  is  known  as  salivary  amylase  or 
ptyalin.     This  is  an  amylolytic  enzyme  (see  page  4),  so  called  because  it 

^  AUaria:  Monalsschr  fiir  Kinderhcilkmide,  to,  179,  igii. 

''Lothrop  and  Gies:  Journal  of  lite  Allied  {Dental)  Societies,  6,  65,  19U. 

'  Id.:  Ibid.,  5,  No.  4,  1910. 


56  PHYSIOLOGICAL    CHEMISTRY 

possesses  the  property  of  transforming  complex  carbohydrates  such  as 
starch  and  dextrin  into  simpler  bodies.  The  action  of  salivary  amylase 
is  one  of  hydrolysis  and  through  this  action  a  series  of  simpler  bodies  are 
formed  from  the  complex  starch.  The  first  product  of  the  action  of  the 
ptyalin  of  the  sahva  upon  starch  paste  is  soluble  starch  (amidulin)  and  its 
formation  is  indicated  by  the  disappearance  of  the  opalescence  of  the 
starch  solution.  This  body  resembles  true  starch  in  giving  a  blue  color 
with  iodine.  Next  follows  the  formation,  in  succession,  of  a  series  of 
dextrins,  called  erythro-dextrin,  a-achroo-dextrin,  ^-achroo-dextrin,  and 
y-achroo-dextrin,  the  erythro-dextrin  being  formed  directly  from  soluble 
starch  and  later  being  itself  transformed  into  a-achroo-dextrin  from  which 
in  turn  are  produced  j3-achroo-dextrin,  y-achroo-dextrin  and  perhaps  other 
dextrins.  Accompanying  each  dextrin  a  small  amount  of  maltose  is 
formed,  the  quantity  of  maltose  growing  gradually  larger  as  the  proc- 
ess of  transformation  progresses.  (Erythro-dextrin  gives  a  red  color 
with  iodine,  the  other  dextrins  give  no  color.)  The  next  stage  is  the 
transformation  of  the  final  dextrin  into  maltose,  the  latter  being  the  prin- 
cipal end-product  of  the  salivary  digestion  of  starch.  At  this  point 
a  small  amount  of  glucose  is  formed  from  the  maltose  through  the  ac- 
tion of  the  enzyme  maltase.  The  above  changes  may  be  represented 
graphically  as  follows: 

Starch 

I 

Soluble  starch 


Erythro-dextrin  Maltose 


a-Achroo-dextrin  Maltose 

I 


l3-Achroo-dextrin  Maltose 


7-Achroo-dextrin  Maltose 

I I 

I 

Maltose 

Salivary  amylase  acts  in  alkaline,  neutral,  or  combined  acid  solu- 
tions. It  will  act  in  the  presence  of  relatively  strong  combined  HCl  (see 
page  140),  whereas  a  trace  (0.003  P^^^  cent  to  0.0006  per  cent)  of  ordinary 


SALIVARY   DIGESTION  57 

free  hydrochloric  acid  will  not  only  prevent  the  action  but  will  destroy 
the  enzyme.  By  sufficiently  increasing  the  alkalinity  of  the  saliva  to 
litmus,  the  action  of  the  salivary  amylase  is  inhibited. 

It  has  been  shown  by  Cannon  that  salivary  digestion  may  proceed 
for  a  considerable  period  after  the  food  reaches  the  stomach,  owing 
to  the  slowness  with  which  the  contents  are  thoroughly  mixed  with 
the  acid  gastric  juice  and  the  consequent  tardy  destruction  of  the 
enzyme.  Food  in  the  pyloric  end  of  the  stomach  is  soon  mixed  with  the 
gastric  secretion,  but  food  in  the  cardiac  end  is  not  mixed  with  the  acid 
gastric  juice  for  a  considerable  period  of  time,  and  in  this  region  during 
that  time  sahvary  digestion  may  proceed  undisturbed. 

It  has  been  found  that  salivary  amylase  acts  more  efficiently  when 
the  saliva  is  diluted  from  4  to  7  times. '^ 

Water  softened  by  lime-  inhibits  the  action  of  salivary  amylase  due 
to  the  presence  of  magnesium  hydroxide  in  this  water. ^  Electrolytes 
have  an  important  influence  upon  the  action  of  amjdases.  The  CI  ion 
has  a  pronounced  facilitating  action  (see  Pancreatic  Amylase). 

The  question  of  the  adaptation  of  the  salivary  secretion  to  diet  is  one 
which  has  received  considerable  attention  in  recent  years.  It  has  been 
claimed,  on  the  basis  of  experimental  evidence/  that  the  continued 
feeding  of  a  carbohydrate  diet  causes  the  secretion  of  a  saliva  which 
contains  a  higher  concentration  of  salivary  amylase  and  one  which  is 
therefore  able  to  more  efficiently  digest  the  carbohydrate  fed.  On  the 
other  hand,  strong  evidence^  has  been  submitted  that  the  amylase  con- 
tent of  the  saliva  is  not  increased  through  the  continued  feeding  of  a 
carbohydrate  diet.  In  general  the  consensus  of  opinion  is  opposed 
to  the  adaptation  of  digestive  secretions  to  diet. 

Maltose,  sometimes  called  glucase,  is  the  second  enzyme  of  the  saliva. 
The  principal  function  of  maltase  is  the  splitting  of  maltose  into  glucose. 
Besides  occurring  in  the  saliva  it  is  also  present  in  the  pancreatic  and 
intestinal  juices.  For  experimental  purposes  the  enzyme  is  ordinarily 
prepared  from  corn.  The  principles  of  the  "reversibility"  of  enzyme 
action  were  first  demonstrated  in  connection  with  maltase  by  Croft  Hill. 

It  is  claimed  that  the  saliva  contains  dipeptide-  and  tripeptidc- 
spHtting   enzymes.^    Leucyl-glycyl-alanine   was    the    tripeptide  split, 

^  Bergeim  and  Hawk:  Jour.  Am.  Clicm.  Soc,  35,  461,  1913. 

^  Prepared  by  treating  tap  water  with  one-sixth  its  volume  of  saturated  lime  water, 
allowing  to  stand  24  hours  and  filtering. 

'  Bergeim  and  Hawk:  Jottr.  Am.  Chem.  Soc,  35,  1049,  1913. 

*  Neilson  and  Terry:  American  Journal  of  Physiology,  15,406,1905;  Neilson  and  Lewis: 
Journal  of  Biological  Chemistry,  4,  501,  1908. 

^  Mendel:  American  Journal  of  the  Medical  Sciences,  Oct.,  1909;  Mendel  and  UnderhiU: 
Journal  of  Biological  Chemistry,  3,  135,  1907;  Mendel,  Chapman  and  Blood:  Medical 
Record,  Aug.  27,  igio. 

8  Koelker:  Zeitschrift  fur  physiol.  Chem.,  76,  27,  191 1, 


58  PHYSIOLOGICAL   CHEMISTRY 

whereas  the  cleavage  of  several  dipeptids  was  brought  about.  The 
action  is  similar  to  that  of  intestinal  erepsin  (see  Chapter  XI).  Later 
investigations  (see  page  199),  apparently  have  demonstrated  that  the 
peptoly  tic  power  of  saliva,  at  least  in  some  cases,  is  due  to  bacteria. 

Microscopical  examination  of  the  saliva  reveals  salivary  corpuscles, 
bacteria,  food  debris,  epithelial  cells,  mucus,  and  fungi.  In  certain 
pathological  conditions  of  the  mouth,  pus  cells  and  blood  corpuscles 
may  be  found  in  the  saliva. 

Experiments  on  Saliva 

A  satisfactory  method  of  obtaining  the  saliva  necessary  for  the  ex- 
periments which  follow  is  to  chew  a  small  piece  of  pure  paraffin  wax, 
thus  stimulating  the  flow  of  the  secretion,  which  may  be  collected  in  a 
small  beaker.  Filtered  saliva  is  to  be  used  in  every  experiment  except 
for  the  microscopical  examination. 

I.  Microscopical  Examination. — Examine  a  drop  of  unfiltered  saliva  micro- 
scopically, after  staining  with  methylene  blue,  and  compare  with  Fig.  19  below. 


Fig.  20. — Microscopical  Constituents  of  Saliva. 

a,  Epithelial  cells;  b,  salivary  corpuscles;  c,  fat  drops;  d,  leucocytes;  e,  f  and  g,  bacteria; 

h,  i,  and  k,  fission-fungi. 

2.  Reaction. — ^Test  the  reaction  to  litmus,  phenolphthalein  and  Congo  red. 

3.  Specific  Gravity. — Partially  fill  a  urinometer  cylinder  with  saliva,  introduce 
the  urinometer,  and  observe  the  reading. 

4.  Test  for  Mucin.— To  a  small  amount  of  saliva  in  a  test-tube  add  1-2  drops 
of  dilute  acetic  acid.     Mucin  is  precipitated. 

5.  Biuret  Test.^ — ^Render  a  Uttle  saliva  alkaline  with  an  equal  volmne  of  KOH 
and  add  a  few  drops  of  a  very  dilute  (2-5  drops  in  a  test-tube  of  water)  copper 
sulphate  solution.    The  formation  of  a  purplish-violet  color  is  due  to  mucin. 

This  reaction  is  given  by  protein  material  and  simply  indicates  that  mucin  is 
a  protein. 

6.  Millon's  Reaction. 2 — Add  a  few  drops  of  Millon's  reagent  to  a  little  saliva. 
A  light  yellow  precipitate  formed  by  the  mucin  gradually  turns  red  upon  being 
gently  heated. 

This  reaction  indicates  the  presence  of  protein  (mucin). 

^  The  significance  of  this  reaction  is  pointed  out  on  p.  98. 
*  The  significance  of  this  reaction  is  pointed  out  on  p.  97. 


I 


SALIVARY   DIGESTION  59 

7.  Preparation  of  Mucin. — Pour  25  c.c.  of  saliva  into  100  c.c.  of  95  per  cent 
alcohol,  stirring  constantly.  Cover  the  vessel  and  allow  the  precipitate  to  stand 
at  least  12  hours.  Pour  off  the  supernatant  liquid,  collect  the  precipitate  on  a 
filter  and  wash  it,  in  turn,  with  alcohol  and  ether.  Finally  dry  the  precipitate, 
remove  it  from  the  paper  and  make  the  following  tests  on  the  mucin :  faj  Test 
its  solubiUty  in  the  ordinary  solvents  (see  page  21);  (b)  Millon's  reaction;  (c) 
dissolve  a  small  amount  in  KOH,  and  try  the  biuret  test  on  the  solution;  (d)  boil 
the  remainder,  with  10-25  c.c.  of  water  to  which  5  c.c.  of  dilute  HCl  has  been 
added,  until  the  solution  becomes  brownish.  Cool,  render  alkaline  with  solid 
KOH,  and  test  by  Fehling's  solution.     A  reduction  should  take  place. 

Mucin  is  what  is  known  as  a  conjugated  protein  or  glycoprotein 
(see  page  112)  and  upon  boiling  with  the  acid  the  carbohydrate  group 
in  the  molecule  has  been  split  off  from  the  protein  portion  and  its 
presence  is  indicated  by  the  reduction  of  Fehling's  solution. 

8.  Inorganic  Matter.- — Test  for  chlorides,  phosphates,  sulphates,  and  cal- 
cium. For  chlorides,  acidify  with  HNO3  and  add  AgNOs.  For  phosphates, 
acidify  with  HNO3,  heat  and  add  molybdate  solution.  ^  For  sulphates,  acidify 
with  HCl  and  add  BaCl2  and  warm.  For  calcium,  acidify  with  acetic  acid,  CH3- 
COOH,  and  add  ammonium  oxalate,  (NH4)2C204. 

9.  Viscosity  Test. — Place  filter  papers  in  two  funnels,  and  to  each  add  an  equal 
quantity  of  starch  paste  (5  c.c).  Add  a  few  drops  of  saUva  to  one  lot  of  paste  and 
an  equivalent  amount  of  water  to  the  other.  Note  the  progress  of  filtration  in 
each  case.     Why  does  one  solution  filter  more  rapidly  than  the  other? 

10.  Test  for  Nitrites. — Add  1-2  drops  of  dilute  H2SO4  to  a  little  saliva  and 
thoroughly  stir.  Now  add  a  few  drops  of  a  potassium  iodide  solution  and  some 
starch  paste.  Nitrous  acid  is  formed  which  liberates  iodine,  causing  the  formation 
of  the  blue  iodide  of  starch. 

11.  Thiocyanate  Tests. — (a)  Ferric  Chloride  Test. — To  a  little  sahva  in  a 
small  porcelain  crucible,  or  dish,  add  a  few  drops  of  dilute  ferric  chloride  and 
acidify  slightly  with  HCl.  Red  ferric  thiocyanate  Fe(SCN)3  forms.  To  show 
that  the  red  coloration  is  not  due  to  iron  phosphate  add  a  drop  of  HgCl2  when 
colorless  mercuric  thiocyanate  forms. 

{b)  Solera's  Reaction. — This  test  depends  upon  the  Uberation  of  iodine  through 
the  action  of  thiocyanate  upon  iodic  acid.  Moisten  a  strip  of  starch  pastc-iodic  acid 
test  paper-  with  a  little  saliva.  If  thiocyanate  be  present  the  test  paper  will  assume 
a  blue  color,  due  to  the  liberation  of  iodine  and  the  subsequent  formation  of  the  so- 
called  iodide  of  starch. 

12.  Digestion  of  Starch  Paste. — To  25  c.c.  of  starch  paste  in  a  small  beaker, 
add  5  drops  of  saliva  and  stir  thoroughly.  At  intervals  of  a  minute  remove  a 
drop  of  the  solution  to  one  of  the  depressions  in  a  test-tablet  and  test  by  the  io- 
dine test.  If  the  blue  color  with  iodine  still  forms  after  five  minutes,  add  another 
5  drops  of  saUva.  The  opalescence  of  the  starch  solution  should  soon  disappear, 
indicating  the  formation  of  soluble  starch  which  gives  a  blue  color  with  iodine. 

^  See  "Reagents  and  Solutions." 

^  This  test  paper  is  prepared  as  follows:  Saturate  a  good  quality  of  filter  paper  with  0.5 
per  cent  starch  paste  to  which  has  been  added  sufficient  iodic  acid  to  make  a  i  per  cent 
solution  of  iodic  acid  and  allow  the  paper  to  dry  in  the  air.  Cut  it  in  strips  of  suitable  size 
and  preserve  for  use. 


6o  PHYSIOLOGICAL    CHEMISTRY 

This  body  should  soon  be  transformed  into  erythro-dextrin  which  gives  a  red 
color  with  iodine,  and  this  in  turn  should  pass  into  achroo-dextrin  which  gives  no 
color  with  iodine.  This  is  called  the  achromic  point.  When  this  point  is  reached 
test  by  Fehling's  test  to  show  the  production  of  a  reducing  body.  A  positive 
Fehling's  test  may  be  obtained  while  the  solution  still  reacts  red  with  iodine 
inasmuch  as  some  maltose  is  formed  from  the  soluble  starch  coincidently  with 
the  formation  of  the  erythro-dextrin.  How  long  did  it  take  for  a  complete  trans- 
formation of  the  starch?  For  a  graphic  representation  of  the  above  changes  see 
page  56. 

13.  Separation  of  the  Products  of  Salivary  Digestion. — To  25  c.c.  of  starch 
paste  in  a  small  beaker  add  i  c.c.  of  saliva  and  stir  thoroughly.  At  intervals  of 
one  minute  test  a  drop  of  the  mixture  by  the  iodine  test.  If  the  blue  color  per- 
sists after  five  minutes  add  another  i  c.c.  of  saUva.  When  the  mixture  reacts 
red  with  iodine,  indicating  that  erythrodextrin  has  been  formed,  add  100  c.c.  of 
95  per  cent  alcohol.  Allow  to  stand  until  the  white  precipitate  has  settled. 
Filter,  evaporate  the  filtrate  to  dryness,  dissolve  the  residue  in  5-10  c.c.  of  water 
and  try  Fehling's  test  (page  26)  and  the  phenylhydrazine  reaction  (see  Glucose,  3, 
page  22).  On  the  dextrin  precipitate  try  the  iodine  test  (page  45).  Also 
hydrolyze  the  dextrin  as  given  under  Dextrin,  4,  page  48. 

14.  Digestion  of  Dry  Starch. — In  a  test-tube  shake  up  a  small  amount  of  dry 
starch  with  a  little  water.  Add  a  few  drops  of  saUva,  mix  well,  and  allow  to 
stand.  After  10-20  minutes  filter  and  test  the  filtrate  by  FehUng's  test.  What 
is  the  result  and  why? 

Dry  starch  is  very  slowly  digested  by  salivary  amylase. 

15.  Digestion  of  Inulin.— To  5  c.c.  of  inulin  solution  in  a  test-tube  add  10  drops 
of  saliva  and  place  the  tube  in  the  incubator  or  water-bath  at  4o°C.  After  one- 
half  hour  test  the  solution  by  Fehling's  test.^  Is  any  reducing  substance  present? 
What  do  you  conclude  regarding  the  salivar>^  digestion  of  inulin? 

16.  Influence  of  Temperature. — In  each  of  four  tubes  place  about  5  c.c.  of 
starch  paste.  Immerse  one  tube  in  cold  water  from  the  faucet,  keep  a  second  at 
room  temperature,  and  place  a  third  in  the  incubator  or  the  water-bath  at  40°C. 
(If  the  temperature  of  the  bath  or  incubator  is  allowed  to  rise  to  7o°C.  or  over  the 
enzyme  is  destroyed  and  no  digestion  takes  place.)  Now  add  to  the  contents  of 
each  of  these  three  tubes  two  drops  of  saliva  and  shake  well;  to  the  contents  of 
the  fourth  tube  add  two  drops  of  boiled  saUva.  Test  frequently  by  the  iodine 
test,  using  the  test-tablet,  and  note  in  which  tube  the  most  rapid  digestion  occurs. 
Explain  the  results. 

17.  Influence  of  Dilution.- — Take  a  series  of  six  test-tubes  each  containing 
9  c.c.  of  water.  Add  i  c.c.  of  saUva  to  tube  i  and  shake  thoroughly.  Remove 
I  c.c.  of  the  solution  from  tube  i  to  tube  2  and  after  mixing  thoroughly  remove 
I  c.c.  from  tube  2  to  tube  3.  Continue  in  this  manner  until  you  have  6  saliva 
solutions  of  gradually  decreasing  strength.  Now  add  starch  paste  in  equal 
amounts  to  each  tube,  ttiiy  very  thoroughly,  and  place  in  the  incubator  or  water- 

1  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the  saliva  it  will  be 
necessary  to  determine  the  extent  of  the  original  reduction  by  means  of  a  "check"  test  (see 

p.  47)- 

'^  The  technic  of  Wohlgemuth's  method  (see  Chapter  X)  may  be  employed  in  this  test 
if  so  desired. 


SALIVARY    DIGESTIOX  6 1 

bath  at  40'C.     After  10-20  minutes  test  by  both  the  iodine  and  Fehhng's  tests. 
In  how  great  dilution  does  your  saliva  act? 

18.  Influence  of  Acids  and  Alkalis. — (a)  Influence  of  Free  Acid. — Prepare  a 
series  of  six  tubes  in  each  of  which  is  placed  4  c.c.  of  one  of  the  following  strengths 
of  free  HCl :  0.2  per  cent,  o.i  per  cent,  0.05  per  cent,  0.025  per  cent,  0.0125  per 
cent  and  0.006  per  cent.  Now  add  2  c.c.  of  starch  paste  to  each  tube  and  shake 
them  thoroughly.  Complete  the  solutions  by  adding  2  c.c.  of  saUva  to  each  and 
repeat  the  shaking.  The  total  acidity  of  this  series  would  be  as  follows :  o.i  per 
cent,  0.05  per  cent,  0.025  per  cent,  0.0125  P^r  cent,  0.006  per  cent  and  0.003  P^r 
cent.  Place  these  tubes  on  the  water-bath  at  40°C.  for  10  20  minutes.  Divide 
the  contents  of  each  tube  into  two  parts,  testing  one  part  by  the  iodine  test  and 
testing  the  other,  after  neutraUzation,  by  Fehhng's  test.     What  do  you  find? 

(b,)  Influence  of  Combined  Acid  (Protein  Salti.  -Repeat  the  first  three  ex- 
periments of  the  above  series  using  combined  hydrochloric  acid  (see  page  140) 
instead  of  the  free  acid.  How  does  the  action  of  the  combined  acid  differ  from 
that  of  the  free  acid?      (For  a  discussion  of  combined  acid  see  page  140. 1 

(c)  Influence  of  AlkaU— Repeat  the  first  four  experiments  under  (ai  replac- 
ing the  HCl  by  2  per  cent,  i  per  cent,  0.5  per  cent  and  0.25  per  cent  NajCOs. 
Neutralize  the  alkalinity  before  trying  the  iodine  test  (see  Starch,  5,  page  45), 

(d)  Nature  of  the  Action  of  Acid  and  AlkaU. — Place  2  c.c.  of  sahva  and  2  c.c, 
of  0.2  per  cent  HCl  in  a  test-tube  and  leave  for  15  minutes.  NeutraUze  the 
solution,  add  4  c.c.  of  starch  paste  and  place  the  tube  in  the  incubator  or  water- 
bath  at  40'C.  In  10  minutes  test  by  the  iodine  and  Fehhng's  tests  and  explain 
the  result.  Repeat  the  experiment,  replacing  the  0.2  per  cent  HCl  by  2  per  cent 
NaoCOs.     What  do  you  deduce  from  these  two  experiments? 

19.  Influence  of  MetaUic  Salts,  etc. — In  each  of  a  series  of  tubes  place  4  c.c. 
of  starch  paste  and  J^  c.c.  of  one  of  the  solutions  named  below.  Shake  well,  add 
^2  c.c.  of  saliva  to  each  tube,  thoroughly  mix,  arid  place  in  the  incubator  or  water- 
bath  at  40°C.  for  10-20  minutes.  Show  the  progress  of  digestion  by  means  of  the 
iodine  and  Fehling  tests.  Use  the  following  chemicals:  MetaUic  sails,  10  per  cent 
lead  acetate,  2  per  cent  copper  sulphate,  5  per  cent  ferric  chloride,  8  per  cent  mer- 
curic chloride;  Neutral  salts,  10  per  cent  sodium  chloride,  10  per  cent  magnesium 
sulphate,  3  per  cent  barium  chloride,  10  per  cent  Rochelle  salt.  Also  tr\'  the  influ- 
ence of  2  per  cent  carbolic  acid,  95  per  cent  alcohol,  and  ether  and  chloroform. 
What  are  your  conclusions? 

Antiseptics  do  not  necessarily  inhibil  enzyme  action. 

20.  Excretion  of  Potassium  Iodide. — Ingest  a  small  dose  of  potassiimi  iodide 
(0.2  gram)  contained  in  a  gelatin  capsule,  quickly  rinse  out  the  mouth  with 
water,  and  then  test  the  saUva  at  once  for  iodine.  This  test  should  be  negative. 
Make  additional  tests  for  iodine  at  two-minute  intervals.  The  test  for  iodine  is 
made  as  follows:  Take  i  c.c.  of  NaNO.  and  i  c.c.  of  dilute  HjSOi'  in  a  test- 
tube,  add  a  little  saliva  directly  from  the  mouth,  and  a  small  amount  of  starch 
paste.  The  formation  of  a  blue  color  signifies  that  the  potassium  iodide  is  being 
excreted  through  the  salivary  glands.  Note  the  length  of  time  elapsing  between 
the  ingestion  of  the  potassium  iodide  and  the  appearance  of  the  first  traces  of  the 
substance  in  the  saliva.     If  convenient,  the  urine  may  also  be  tested.     The 

'  Instead  of  this  mixture  a  lew  drops  of  HXO3  possessing  a  yellowish  or  brownish  color 
due  to  the  presence  of  HXO.;  may  be  employed. 


62  PHYSIOLOGICAL   CHEMISTRY 

chemical  reactions  taking  place  in  this  experiment  are  indicated  in  the  following 
equations : 

(a)  2NaN02+H2S04-^2HN02+Na2S04. 

(b)  2KI  +  H2S04^2HI  +  K2S04. 

(C)  2HNO2+       2HI-^l2  +  2H20  +  2NO. 


CHAPTER  IV 
PROTEINS  :i  THEIR  DECOMPOSITION  AND  SYNTHESIS 

The  proteins  are  a  class  of  substances,  which  in  the  light  of  our  pres- 
ent knowledge,  consist,  in  the  main,  of  combinations  of  a-amino  acids  or 
their  derivatives.  These  protein  substances  form  the  chief  constituents 
of  many  of  the  fluids  of  the  body,  constitute  the  organic  basis  of  animal 
tissue,  and  at  the  same  time  occupy  a  decidedly  preeminent  position 
among  our  organic  food-stuffs.  They  are  absolutely  necessary  to  the 
uses  of  the  animal  organism  for  the  continuance  of  life  and  they  cannot 
be  satisfactorily  replaced  in  the  diet  of  such  an  organism  by  any  other 
dietary  constituent  either  organic  or  inorganic.  Such  an  organism  may 
exist  without  protein  food  for  a  period  of  time,  the  length  of  the  period 
varying  according  to  the  specific  organism  and  the  nature  of  the  substi- 
tution offered  for  the  protein  portion  of  the  diet.  Such  a  period  is,  how- 
ever, distinctly  one  of  existence  rather  than  one  of  normal  life  and  one 
which  is  consequently  not  accompanied  by  such  a  full  and  free  exercise 
of  the  various  functions  of  the  organism  as  would  be  possible  upon  an 
evenly  balanced  ration,  i.e.,  one  containing  the  requisite  amount  of 
protein  food.  These  protein  substances  are,  furthermore,  essential 
constituents  of  all  living  cells  and  therefore  without  them  vegetable  life 
as  well  as  animal  life  is  impossible. 

The  proteins,  which  constitute  such  an  important  group  of  sub- 
stances, differ  from  carbohydrates  and  fats  very  decidedly  in  elementary 
composition.  In  addition  to  containing  carbon,  hydrogen,  and  oxygen, 
which  are  present  in  fats  and  carbohydrates,  the  proteins  invariably 
contain  nitrogen  in  their  molecule  and  generally  sulphur  also.  Pro- 
teins have  also  been  described  which  contain  phosphorus,  iron,  copper, 
iodine,  manganese,  and  zinc.  The  percentage  composition  of  the  more  im- 
portant members  of  the  group  of  protein  substances  would  fall  within 
the  following  limits:  C  =  50-55  per  cent,  H  =  6-7.3  P^r  cent,  0  = 
19-24  per  cent,  N=  15-19  per  cent,  8  =  0.3-2.5  per  cent,  P  =  o.4- 
0.8  per  cent  ivhen  present.  WHien  iron,  copper,  iodine,  manganese,  or 
zinc  are  present  in  the  protein  molecule  they  are  practically  without 

^  The  term  proleid  has  been  very  widely  used  by  English-speaking  scientists  to  signify 
the  class  of  substances  we  have  called  proteins. 

63 


64  PHYSIOLOGICAL   CHEMISTRY 

exception  present  only  in  traces  and  with  the  exception  of  iodine  are 
probably  not  constituents  of  the  protein  molecule.^ 

Of  all  the  various  elements  of  the  protein  molecule,  nitrogen  is  by  far 
the  most  important.  The  human  body  needs  nitrogen  for  the  continua- 
tion of  life,  but  it  cannot  use  the  nitrogen  of  the  air  or  that  in  various 
other  combinations  as  we  find  it  in  nitrates,  nitrites,  etc.  However,  in 
the  protein  molecule  the  nitrogen  is  present  in  a  form  which  is  utilizable 
by  the  body.  The  nitrogen  in  the  protein  molecule  occurs  in  at  least 
four  different  forms  as  follows : 

I.  Monamino  acid  nitrogen. 
11.  Diamino  acid  nitrogen  or  basic  nitrogen. 

III.  Amide  nitrogen. 

IV.  A  guanidine  residue. 

The  actual  structure  of  the  protein  molecule  is  still  unknown,  and  we 
have  as  yet  no  means  by  which  its  molecular  weight  can  be  even  approxi- 
mately established.  The  many  attempts  which  have  been  made  to 
determine  this  have  led  to  very  different  results,  some  of  which  are  given 
in  the  following  table: 

Globin  =15000—16086 

Oxyhemoglobin  =  14800— 15000  — 16655  — 16730 

Of  these  figures,  those  given  for  oxyhemoglobin  deserve  the  most 
consideration,  for  these  are  based  on  the  atomic  ratios  of  the  sulphur 
and  iron  contained  in  this  substance.  The  simplest  formula  that  can 
be  calculated  from  analyses  of  oxyhemoglobin,  namely, 

C658Hii8lN207S2FeO210 

serves  to  show  the  great  complexity  of  this  substance. 

The  decomposition-  of  protein  substances  may  be  brought  about  by 
oxidation  or  hydrolysis,  but  inasmuch  as  the  hydrolytic  procedure  has 
been  productive  of  the  more  satisfactory  results,  that  type  of  decomposi- 
tion procedure  alone  is  used  at  present.  This  hydrolysis  of  the  protein 
molecule  may  be  accomplished  by  acids,  alkalis,  or  superheated  steam, 
and  in  digestion  by  the  action  of  the  proteolytic  enzymes.  The  char- 
acter of  the  decomposition  products  varies  according  to  the  method 
utilized  in  tearing  the  molecule  apart.  Bearing  this  in  mind,  we  may 
say  that  the  decomposition  products  of  proteins  include  proteoses,  pep- 
tones, peptides,  carbon  dioxide,  ammonia,  hydrogen  sulphide,  and  amino 

1  Some  investigators  regard  these  elements  as  contaminations,  or  constituents  of  some 
non-protein  substance  combined  with  the  protein. 

2  The  terms  "degradation,"  "dissociation,"  and  "cleavage,"  are  often  used  in  this  con- 
nection. 


PROTEINS  65 

acids.  These  amino  acids'  constitute  a  long  list  of  important  substances 
which  contain  nuclei  belonging  either  to  the  aliphatic,  carbocyclic,  or 
heterocyclic  series.  The  list  includes  glycocoll,  alanine,  serine,  phenyl- 
alanine, tyrosine,  cystine,  tryptophane,  histidine,  valine,  arginine,  leucine, 
isoleucine,  lysine,  aspartic  acid,  glutamic  acid,  proline,  oxyproline,  and 
diaminotrihydroxydodecanoic  acid.  Of  these  amino  acids,  tyrosine  and 
phenylalanine  contain  carbocyclic  nuclei:  histidine,  proline,  and  trypto- 
phane contain  heterocyclic  nuclei:  and  the  remaining  members  of  the 
list,  as  given,  contain  aliphatic  nuclei.  The  amino  acids  are  preemi- 
nently the  most  important  class  of  protein  decomposition  products. 
These  amino  acids  are  all  a-amino  acids,  and,  with  the  exception  of 
glycocoll,  are  all  optically  active.  Furthermore,  they  are  amphoteric 
substances  and  consequently  are  able  to  form  salts  with  both  bases  and 
acids.  These  properties  are  inherent  in  the  NH2  and  COOH  groups  of 
the  amino  acids. 

The  decomposition  products  of  protein  may  be  grouped  as  pri- 
mary and  secondary  decomposition  products.  By  primary  products  are 
meant  those  which  exist  as  radicals  within  the  protein  molecule  and 
which  are  hberated,  upon  cleavage  of  this  molecule,  with  their  carbon 
chains  intact  and  the  position  of  their  nitrogen  unaltered.  The  second- 
ary products  are  those  which  result  from  the  disintegration  of  the 
primary  cleavage  products.  No  matter  what  method  is  used  to  de- 
compose a  given  protein  molecule,  the  primary  products  are  largely  the 
same  under  all  conditions.^ 

In  the  process  of  hydrolysis  the  protein  molecule  is  gradually  broken 
down  and  less  complicated  aggregates  than  the  original  molecule  are 
formed,  which  are  known  as  proteoses,  peptones,  and  peptides,  and  which 
still  possess  true  protein  characteristics.  Further  hydrolysis  causes  the 
ultimate  transformation  of  these  substances,  of  a  protein  nature,  into  the 
amino  acids  of  known  chemical  structure.  In  this  decomposition  the 
protein  molecule  is  not  broken  down  in  a  regular  manner  into  }/^,  ^, 
^i  portions  and  the  amino  acids  formed  in  a  group  at  the  termination  of 
the  hydrolysis.  On  the  contrary,  certain  amino  acids  are  formed  very 
early  in  the  process,  in  fact  while  the  main  hydrolytic  action  has  pro- 
ceeded no  further  than  the  proteose  stage.  Gradually  the  complexity 
of  the  protein  portion  undergoing  decomposition  is  simplified  by  the 
splitting  off  of  the  amino  acids  and  finally  it  is  so  far  decomposed 
through  previous  cleavages  that  it  yields  only  amino  acids  at  the 
succeeding  cleavage.  In  short,  the  general  plan  of  the  hydrolysis  of 
the  protein  molecule  is  similar  to  the  hydrolysis  of  starch.     In  the  case 

^For  a  discussion  of  amino  acids  see  Underhill's  "Plij'siology  of  Amino  Acids,''  Yale 
University  Press,  Nov.,  1915. 

*  Alkaline  hydrolysis  yields  urea  and  brrttVAtwc  which  result  iiom  arginine,  the  product  of 
acid  hj'drolysis. 


66  PHYSIOLOGICAL   CHEMISTRY 

of  starch  there  is  formed  a  series  of  dextrins  of  gradually  decreasing 
complexity  and  coincidently  with  the  formation  of  each  dextrin  a  small 
amount  of  sugar  is  split  off  and  finally  nothing  but  sugar  remains.  In 
the  case  of  protein  hydrolysis  there  is  a  series  of  proteins  of  gradually 
decreasing  complexity  produced  and  coincidently  with  the  formation  of 
each  new  protein  substance  amino  acids  are  split  oft"  and  finally  the  sole 
products  remaining  are  amino  acids. 

Inasmuch  as  diversity  in  the  method  of  decomposing  a  given  protein 
does  not  result  in  an  equally  diversified  line  of  decomposition  products, 
but,  on  the  other  hand,  yields  products  which  are  quite  comparable  in 
character,  it  may  be  argued  that  there  are  probably  well-defined  Hnes  of 
cleavage  in  the  individual  protein  molecule  and  that  no  matter  what  the 
force  brought  to  bear  to  tear  such  a  molecule  apart,  the  disintegration, 
when  it  comes,  will  yield  in  every  case  certain  definite  fragments. 
These  fragments  may  be  called  the  "building  stones"  of  the  protein 
molecule,  a  term  used  by  some  of  the  German  investigators.  Take,  for 
example,  the  decomposition  of  protein  which  may  be  brought  about 
through  the  action  of  the  enzyme  trypsin  of  the  pancreatic  juice. 
When  this  enzyme  is  allowed  to  act  upon  a  given  protein,  the  latter  is 
disintegrated  in  a  series  of  definite  cleavages,  resulting  in  the  formation 
of  proteoses,  peptones,  and  peptides  in  regular  order,  the  peptides  being 
the  last  of  the  decomposition  products  which  possess  protein  character- 
istics. They  are  all  built  up  from  amino  acids  and  are  therefore  closely 
related  to  these  acids  on  the  one  side  and  to  peptones  on  the  other. 
We  have  di-,  tri-,  tetra-,  penta-,  dec  a-,  and  poly-peptides  which  are 
named  according  to  the  number  of  amino  acids  included  in  the  peptide 
molecule.  Following  the  peptides  there  are  a  diverse  assortment  of 
monamino  and  diamino  acids  which  constitute  the  final  products  of 
the  protein  decomposition.  These  acids  are  devoid  of  any  protein 
characteristics  and  are  therefore  decidedly  different  from  the  original 
substance  from  which  they  were  derived.  From  a  protein  of  huge 
molecular  weight,  a  typical  colloid,  perhaps  but  slightly  soluble,  and 
entirely  non-dift'usible,  we  have  passed  by  way  of  proteoses,  peptones, 
and  peptides  to  a  class  of  simpler  crystalline  substances  which  are,  for 
the  most  part,  readily  soluble  and  diffusible. 

These  amino  acids  after  their  production  in  the  process  of  digestion, 
as  just  indicated,  are  synthesized  within  the  cells  of  the  organism  to 
form  protein  material  which  goes  to  build  up  the  tissues  of  the  body. 
It  is  thus  seen  that  the  amino  acids  are  of  prime  importance  in  the 
animal  economy.  It  was  formerly  believed  that  these  essential  factors 
in  metabolism  and  nutrition  could  not  be  produced  within  the  animal 
organism  from  their  elements,  but  were  only  yielded  upon  the  hydrol- 


PROTEINS 


67 


ysis  of  ingested  protein  of  animal  or  vegetable  origin.  Experi- 
ments, however,  by  Abderhalden  and  by  Grafe  and  Schlapfer  and 
others  indicate  that  the  nitrogen  of  food  protein  may  in  part  be  re- 
placed by  ammonium  salts.  Experiments  by  Osborne  and  others 
also  indicate  amino  acid  synthesis  by  animals. 

Important  data  regarding  the  decomposition  products  of  the  protein 
molecule  are  given  in  the  tables  which  follow. 


COMPARISON  OF  THE  DECOMPOSITION  PRODUCTS  OF  PROTAMINES, 

AND 

OTHER  PROTEINS. 

Protamines.! 

Other  Proteins. 

(Per  cent  of  total  nitrogen  of 

(Per  cent  of  amino  acids  in 

amino  acid.) 

proteins.) 

Decomposition 

o5 
a 
■c 

6 

G    . 

•c 

c 

a 
'a 

V 

c 

■5 

6 

c 

"d 

'c 

i 

d 

"d 

Product. 

.a 
B 

1 

0 
3 

u 

6 

a 

u 

0 

E 

.2.S 

XI 

0 

0 

13 
0 

5 

• 

GlycocoU .... 

1 

0 

3-8 

0 

16. S 

0 

0 

Alanine . 


3.6 


1.5      0.8      4.2      9.79 


Valine. 


+    I    +    I    +    |l.6s|   3-34 


6.2 


7.2 


Leucine I    + 


14-5 


9.4       2.1    :29.o     19. SS 


Proline I  3- 


+    4.3 


4.1      I   6.7       S-2    !    2.3       9-04 


Phenylalanine. 


2.3s 


6.SS 


Aspartic  acid 0.58 


4-S 


1.4      0.56.   4.4      1. 71 


Glutamic  acid ■ 43-66   I18.74   !ii-0       l.88j    1.7     26.17 


Serine. 


+    3-25    0.13       0.33   I   o.s      0.4      0.6 


Tyrosine. 


i-s: 


45 


1.3      3.SS 


Arginine 88.8  67  .7J63.5    8.7:28.0  88.0  89.2 j   3.16   I14.2         4.84    7-62}   5-4   '    ^SS 


Lysine 

1  8.4  30.3!  6.6  .  .  .  . 

. . . .     0 

;v 

S-95    2.7S|   4.3 

0 

Histidine 

II. 8 

. . . .  0.61 

2.2 

2.50    0.40  II. 0 

0.82 

Tryptophane 

....|    + 

+ 

i.. ..!....' 

+ 

1.5          0         + 

0 

. . . . '  0 .45 

1 .00 

0.06s      0        0.3 

? 

? 

2.0 

1   0.23    6.4 

I  .0 

? 

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'0.75     •  •    • 

? 

decanoic  acid.                   '          '          1                              1 

5.33 

2.3 

i.6i     

3  64 

i     1     1     I    ■ 

When  we  examine  the  formulas  of  the  principal  members  of  the 
crystalline  end-products  of  protein  decomposition  we  note  that  they  are 

■  ''Kossel:  Zeit.  physiol.  Chem.,  44,  347,  1905. 
^Osborne  and  Guest:  Jour.  Biol.  Chcm.,  0,  425,  19 11. 
^.\bderhalden,  Kossel  and  others. 

*  Abderhalden,  Fischer,  Morner  and  others. 

'Fischer,  Levene  and  Aders:  Zeil.  physiol.  Chem.,  35,  70,   1902;  also    Lcvene     and 
Beatty:  Ibid.,  49,  252,  1906. 

"Abderhalden:  Zcit.  physiol.  Chem.,  37,  4S4,  1903. 
'Osborne  and  Liddle,  .\m.  Jour.  Physiol.,  26,  295,  1910. 

*  This  unique  and  important  protein  has  probably  been    more  carefully    analyzed 
than  any  other. 


68 


PHYSIOLOGICAL    CHEMISTRY 


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PROTEINS 


69 


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yo  PHYSIOLOGICAL    CHEMISTRY 

invariably  acids,  as  has  already  been  mentioned,  and  contain  an  NH2 
group  in  the  a  position.  This  relation  of  the  NHo  group  to  the  acid  radi- 
cal is  constant,  no  matter  what  other  groups  or  radicals  are  present.  We 
may  have  straight  chains  as  in  alanine  and  glutamic  acid,  the  benzene 
ring  as  in  phenylalanine,  or  we  may  have  sulphurized  bodies  as  in  cystine 
and  still  the  formula  is  always  of  the  same  type,  i.e., 

NH2 

R— CH— COOH 

• 

It  is  seen  that  this  characteristic  grouping  in  the  amino  acid  provides 
each  one  of  these  ultimate  fragments  of  the  protein  molecule  with  both  a 
strong  acid  and  a  strong  basic  group.  For  this  reason  it  is  theoretically 
possible  for  a  large  number  of  these  amino  acids  to  combine  and  the  re- 
sulting combinations  may  be  very  great  in  number,  since  there  is  such 
a  varied  assortment  of  the  acids.  The  protein  molecule,  which  is  of 
such  mammoth  proportions,  is  probably  constructed  on  a  foundation  of 
this  sort.  Much  valuable  data  have  been  collected  regarding  the  syn- 
thetic production  of  protein  substances,  the  leaders  in  this  line  of  in- 
vestigation being  Fischer  and  Abderhalden.  After  having  gathered  a 
mass  of  data  regarding  the  final  products  of  the  protein  decomposition 
and  demonstrating  that  amino  acids  were  the  ultimate  results  of  the 
various  forms  of  decomposition,  these  investigators,  and  notably 
Fischer,  set  about  in  an  effort  to  form,  from  these  amino  acids,  by 
synthetic  means,  substances  which  should  possess  protein  character- 
istics. The  simplest  of  these  bodies  formed  in  this  way  was  synthesized 
from  two  molecules  of  glycocoll  with  the  liberation  of  water,  thus: 


CH2-(NH2)-CO  OH  H  HN-CH2-COOH. 

The  body  thus  formed  is  a  dipeptide,  called  glycyl-glycine.  In  an  analo- 
gous manner  may  be  produced  leucyl-leucine,  through  the  synthesis  of 
two  molecules  of  leucine  or  leucyl-alanyl-glycine  through  the  union  of  one 
molecule  of  leucine,  one  of  alanine,  and  one  of  glycocoll.  By  this  pro- 
cedure Fischer  and  his  pupils  have  been  able  to  make  a  large  number  of 
peptides  containing  varied  numbers  of  amino  acid  radicals,  the  name 
polypeptides  being  given  to  the  whole  group  of  synthetic  substances  thus 
formed.  One  of  the  most  complex  polypeptides  yet  produced  is  one 
containing  fifteen  glycocoll  and  three  leucine  residues. 

Notwithstanding  the  fact  that  most  synthetic  polypeptides  are  pro- 
duced through  a  union  of  amino  acids  by  means  of  their  imide  bonds,  it 
must  not  be  imagined  that  the  protein  molecule  is  constructed  from 


PROTEINS  7 1 

amino  acids  linked  together  in  straight  chains  in  a  manner  analogous  to 
the  formation  of  simple  peptides,  such  as  glycyl-glycine.  The  molecular 
structure  of  the  proteins  is  much  too  complex  to  be  explained  upon  any 
such  simple  formation  as  that.  There  must  be  a  variety  of  linkings, 
since  there  is  a  varied  assortment  of  decomposition  products  of  totally 
different  structure. 

]\Iany  of  these  synthetic  bodies  respond  to  the  biuret  test,  are  pre- 
cipitated by  phosphotungstic  acid,  and  behave,  in  other  ways,  as  to 
leave  no  doubt  as  to  their  protein  characteristics.  For  instance,  a 
number  of  amino  acids  each  possessing  a  sweet  taste  have  been  syn- 
thesized in  such  a  manner  as  to  yield  a  polypeptide  of  bitter  taste,  a 
well-known  characteristic  of  peptones.  From  the  fact  that  the  poly- 
peptides formed  in  the  manner  indicated  have  free  acidic  and  basic 
radicals  we  gather  the  explanation  of  the  amphoteric  character  of  true 
proteins. 

For  the  benefit  of  those  especially  interested  in  such  matters  a  photo- 
graph of  the  Fischer  apparatus  (Fig.  24,  page  75)  used  in  the  fractional 
distillation,  in  vacuo,  of  the  esters  of  the  decomposition  products  of  the 
proteins,  as  well  as  micro-photographs  and  drawings  of  preparations  of 
several  of  these  decomposition  products  (Figs.  21  to  33,  pages  72  to  84) 
are  introduced.  For  the  preparations  and  the  photograph  of  the  appa- 
ratus the  author  is  indebted  to  Dr.  T.  B.  Osborne,  of  New  Haven,  Conn., 
who  has  made  many  important  observations  upon  the  hydrolysis  of 
proteins.  The  reproduction  of  the  crystalline  form  of  some  of  the  more 
recent  of  the  products  may  be  of  interest  to  those  viewing  the  field  of 
physiological  chemistry  from  other  than  the  student's  aspect. 

An  extended  discussion  of  the  various  decomposition  products  being 
out  of  place  in  a  book  of  this  character,  we  will  simply  make  a  few  general 
statements  in  connection  with  the  primary  decomposition  products. 

DISCUSSION  OF  THE  PRODUCTS 

Ammonia,  XH3. — Ammonia  is  an  important  decomposition  product 
of  all  proteins  and  probably  arises  from  an  amide  group  combined 
with  a  carboxyl  group  of  some  of  the  amino  acids.  It  is  possible  that  the 
dibasic  acids,  aspartic  and  glutamic,  furnish  most  of  these  carboxyl 
groups.  This  is  indicated  by  the  more  or  less  close  relationship  which 
exists  between  the  amount  of  ammonia  and  that  of  the  dibasic  acids 
which  the  several  proteins  yield  upon  decomposition.  The  elimination 
of  the  ammonia  from  proteins  under  the  action  of  acids  and  alkaUs  is 
very  similar  to  that  from  amides  like  asparagine. 

Glycocoll,  CH2-(XH2)-COOH.— Glycocoll,  or  amino  acetic  acid,  is 


72  PHYSIOLOGICAL   CHEMISTRY 

the  simplest  of  the  amino  acids  which  occurs  as  a  protein  decomposition 
product^  and  has  the  following  formula: 

NH2 
H— C— COOH. 

H 

Glycocoll,  as  the  formula  shows,  contains  no  asymmetric  carbon  atom, 
and  is  the  only  amino  acid  yielded  by  protein  decomposition  which  is 
optically  inactive.  Glycocoll  and  leucine  were  among  the  first  decom- 
position products  of  proteins  to  be  discovered.  Upon  administering 
benzoic  acid  to  man  or  lower  animals  the  output  of  hippuric  acid  in  the 


Fig.  21. — ^Glycocoll  Ester  Hydrochloride. 

urine  is  greatly  increased,  thus  showing  a  synthesis  of  benzoic  acid  and 
glycocoll  in  the  organism  (see  page  585,  Chapter  XXVII).  Glycocoll, 
ingested  in  small  amount,  is  excreted  in  the  urine  as  urea,  whereas  if 
administered  in  excess  it  appears  in  part  unchanged  in  the  urine.  It  is 
usually  separated  from  the  mixture  of  protein  decomposition  products 
as  the  hydrochloride  of  the  ester.  The  crystalHne  form  of  this  com- 
pound is  shown  in  Fig.  21. 

Alanine,  CH3-CH(NH2)-COOH.-^Alanine  is  a-amino-propionic  acid, 
and  as  such  it  may  be  represented  structurally  as  follows: 

H    NH2 
H— C— C— COOH. 
H    H 

^  Amino  formic  acid  (carbamic  acid),  NH2COOH,  is  the  simplest  amino  acid. 


PROTEINS 


73 


Obtained  from  protein  substances,  alanine  is  dextro-rotatory,  is  very 
soluble  in  water,  and  possesses  a  sweet  taste.  Tyrosine,  phenylalanine, 
cystine,  and  serine  are  derivatives  of  alanine.  This  amino  acid  has  been 
obtained  from  nearly  all  proteins  examined.  Its  absence  from  those 
proteins  from  which  it  has  not  been  obtained  has  not  been  proven. 
Most  proteins  yield  relatively  small  amounts  of  alanine. 

Serine,  CH2(OH)CH(NH2)-COOH. — Serine  is  a-amino-^-hydroxy- 
propionic  acid  and  possesses  the  following  structural  formula: 

OHNH2 
H— C— C— COOH. 


H    H 

Serine  obtained  from  proteins  is  levo-rotatory,  possesses  a  sweet  taste, 
and  is  quite  soluble  in  water.     Serine  is  not  obtained  in  quantity  from 


Fig.  22. — Serine. 

most  proteins,  but  is  yielded  abundantly  by  silk  glue.     Owing  to  the 

diflSculty  of  separating  serine  it  has  not  been  found  in  a  number  of 

proteins  in  which  it  probably  occurs.     Serine  crystals  are  shown  in  Fig. 

22. 

Phenylalanine,  C6H6CH2-CH(NHo)  COOH.— This  product  is  ^- 

phenyl-a-amino-propionic  acid,  and  may  be  represented  graphically  as 

follows : 

H    NH2 

I       I 
C— C— COOH. 

H    H 


74 


PHYSIOLOGICAL    CHEMISTRY 


The  levo-rotatory  form  is  obtained  from  proteins.  Phenylalanine  has 
been  obtained  from  all  the  proteins  examined  except  from  the  pro- 
tamines and  some  of  the  albuminoids.  The  yield  of  this  body  from  the 
decomposition  of  proteins  is  frequently  greater  than  the  yield  of  tyrosine. 
The  crystalline  form  of  phenylalanine  is  shown  in  Fig.  23. 

Tyrosine,  C6H5(.OH)CH2CH(NH2)COOH.— Tyrosine,  one  of  the 
first  discovered  end-products  of  protein  decomposition,  is  the  amino 
acid,  p-hydroxy-^-phenyl-a-amino-propionic  acid  or  hydroxy  phenylala- 
nine.    It  has  the  following  formula: 

H    NH2 


C— C— COOH. 


H    H 


OH 


The  tyrosine  which  results  from  protein  decomposition  is  usually  levo- 
rotatory.  Tyrosine  is  one  of  the  end-products  of  tryptic  digestion  and 
usually  separates  in  conspicuous  amount  early  in  the  process  of  diges- 


FiG.  23. — Phenylalanine. 


tion.     It  does  not  occur,  however,  as  an  end-product  of  the  decomposi- 
tion of  gelatin. 

Tyrosine  is  found  in  old  cheese,  and  derives  its  name  from  this  fact. 
It  crystallizes  in  tufts,  sheaves,  or  balls  of  fine  needles,  which  decompose 
at  295°C.  and  are  sparingly  soluble  in  cold  (1-2454)  water,  but  much 
more  so  in  boiling  (1-154)  water.  Tyrosine  forms  soluble  salts  with 
alkalis,  ammonia,  or  mineral  acids,  and  is  soluble  with  difficulty  in 
acetic  acid.     It  responds  to  Millon's  reaction,  thus  showing  the  presence 


PROTEINS 


75 


of  the  hydroxyphenyl  group,  but  gives  no  other  protein  test.  The  aro- 
matic groups  present  in  tyrosine,  phenylalanine,  and  tryptophane  cause 
proteins  to  yield  a  positive  xanthoproteic  reaction.  In  severe  cases  of 
typhoid  fever  and  smallpox,  in  acute  yellow  atrophy  of  the  liver,  and  in 


Fig.  24. — Fischer  Apparatus. 

Reproduced  from  a  photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University  of  Penn- 
sylvania.    The  negative  was  furnished  by  Dr.  T.  B.  Osborne,  of  New  Haven,  Conn. 

A,  Tank  into  which  freezing  mixture  is  pumped  and  from  which  it  flows  through  the 
condenser,  B;  C,  flask  from  which  the  esters  are  distilled,  the  distillate  being  collected  in  D; 
E,  a  Dewar  flask  containing  liquid  air  serving  as  a  cooler  for  condensing  tube  F;  G  and  G', 
tubes  leading  to  the  Geryck  pump  by  which  the  vacuum  is  maintained;  /,  tube  leading  to  a 
McLeod  gauge  (not  shown  in  figure);  J,  a  bath  containing  freezing  mixture  in  which  the 
receiver  I)  is  immersed;  A',  a  bath  of  water  during  the  first  part  of  the  distillation  and  of 
oil  during  the  last  part  of  the  process;  1-5,  stop  cocks  which  permit  the  cutting  out  of 
different  parts  of  the  apparatus  as  the  procedure  demands. 


acute  phosphorus  poisoning,    tyrosine   has  been  found  in   the   urine. 
Tyrosine  crystals  are  shown  in  Fig.  25,  page  76. 

Cystine,  C6Hio04NoS2.^Friedmann  has  shown  cystine  to  be 
a-diamino-^-dithiolactyl  acid  and  to  possess  the  following  structural 
formula : 


76 


PHYSIOLOGICAL   CHEMISTRY 

CH2  ■  S — S  •  CH2 

I         I 

CHNH2    CHNH,. 

I  I 

COOH       COOH 


Fig.  25. — Tyrosine. 

Cystine  is  the  principal  sulphur-containing  body  obtained  from  the 
decomposition  of  protein  substances.  It  is  obtained  in  greatest  amount 
as  a  decomposition  product  of  keratin-containing  tissues  as  horn,  hoof, 


Fig.  26. — Cystine. 


and  hair.  Cystine  occurs  in  small  amount  in  normal  urine  and  is 
greatly  increased  in  quantity  under  certain  pathological  conditions.  It 
crystallizes  in  thin,  colorless,  hexagonal  plates  which  are  shown  in  Fig. 


PROTEINS  77 

26.     Cystine  is  very  slightly  soluble  in  water  but  its  salts,  with  both 
bases  and  acids,  are  readily  soluble  in  water.     It  is  levo-rotator}- . 

It  was  formerly  claimed  that  cystine  occurred  in  two  forms,  i.e.^ 
stone-cysdne  and  protein-cystine,  and  that  these  two  forms  are  distinct 
in  their  properties.     This  view  is  incorrect. 

For  the  preparation  of  cystine  from  wool  or  hair  see  page  87. 

For  a  discussion  of  cystine  sediments  in  urine  see  Chapter  XXIV. 

Tryptophane,  C8H6N-CH2CH(NH2)-COOH.— Recently  Ellinger 
and  Flamand  have  shown  that  tryptophane  possesses  the  following 
formula : 

CCH2CH(NH2)COOH 


It  is  therefore  ^-indol-a-amino-propionic  acid.  Tyrophane  is  the 
mot Jier-subs lance  of  indole,  skatole,  skatole  acetic  acid  and  skatole  carhoxylic 
acid,  all  of  which  are  formed  as  secondary  decomposition  products  of 
proteins  (see  Chapter  XIII  on  Putrefaction  Products).  Its  presence 
in  protein  substances  may  be  shown  by  means  of  the  Hopkins-Cole 
reaction  (see  page  98) .  It  may  be  detected  in  a  tryptic  digestion  mix- 
ture through  its  property  of  giving  a  \dolet  color  reaction  with  bromine 
water. ^  Tryptophane  is  \delded  by  nearly  all  proteins,  but  has  been 
shown  to  be  entirely  absent  from  zein,  the  prolamin  (alcohol-soluble  pro- 
tein) of  maize  and  also  from  gelatin. 

According  to  Osborne  and  jMendel,^  tryptophane  is  present  in  maxi- 
mum amount  in  lactalhumin.  Upon  being  heated  to  285°C.  trytophane 
decomposes  with  the  evolution  of  gas. 

Histidine,  C3H3N2-CH2-CH(XH2)-COOH.— Histidine   is  a-amino- 

.^-imidazol-propionic    acid    or   ^-imidazol-alaniuc    with    the    following 

structural  formula: 

H    NH2 

1       I 
HC         C— C— C— COOH. 

I       I 
H    H 
HN        N 

\/ 
CH 

The  histidine  obtained  from  proteins  is  levo-rotatory.  It  has  been 
obtained  from  all  the  proteins  thus  far  examined,  the  majority  of  them 
3'ielding  about  2.5  per  cent  of  the  amino  acid.     However,  about  11  per 

^  Kurajeff:  Zeit.  physiol.  Client.,  36,  501,  1898-99. 

*  Osborne  and  Mendel:  Joitr.  Biol.  Chem.,  20,  357,  1915. 


78  PHYSIOLOGICAL    CHEMISTRY 

cent  was  obtained  by  Abderhalden  from  globin,  the  protein  constituent 
of  oxyhemoglobin,  and  about  13  per  cent  by  Kossel  and  Kutscher  from 
the  protamine  stiirine. 

Crystals  of  histidine  dichloride  are  shown  in  Fig.  27. 

Knoop's  Color  Reaction  for  Histidine. — To  an  aqueous  solution  of  histidine 
or  a  histidine  salt  in  a  test-tube  add  a  little  bromine  water.  A  yellow  coloration 
develops  in  the  cold  and  upon  further  addition  of  bromine  water  becomes  perma- 
nent. If  the  tube  be  heated/  the  color  will  disappear  and  will  shortly  be  re- 
placed by  a  faint  red  coloration  which  gradually  passes  into  a  deep  wine  red. 
Usually  black,  amorphous  particles  separate  out  and  the  solution  becomes 
turbid. 

The  reaction  cannot  be  obtained  in  solutions  containing  free  alkali. 
It  is  best  to  use  such  an  amount  of  bromine  as  will  produce  a  permanent 


■V  C^ 


Fig.  27. — Histidine  Dichloride. 

yellow  color  in  the  cold.  The  use  of  a  less  amount  of  bromine  than  this 
produces  a  weak  coloration,  whereas  an  excess  of  bromine  prevents  the 
reaction.  The  test  is  not  very  delicate,  but  a  characteristic  reaction 
may  always  be  obtained  in  i  :  1000  solutions.  The  only  histidine  de- 
rivative which  yields  a  similar  coloration  is  imidazolethylamine,  and 
the  reaction  in  this  case  is  rather  weak  as  compared  with  the  color  ob- 
tained with  histidine  or  histidine  salts. 

Valine,  C5H11NO2. — The  amino-valerianic  acid  obtained  from 
proteins  is  a-amino-isovalerianic  acid,  and  as  such  bears  the  following 
formula : 

CH3    NH2 

H— C C— COOH. 

CH3    H 

^  The  same  reaction  will  take  place  in  the  cold  more  slowly. 


PROTEINS  79 

It  closely  resembles  leucine  in  many  of  its  properties,  but  is  more  soluble 
in  water.  It  is  a  difficult  matter  to  identify  valine  in  the  presence  of 
leucine  and  isoleucine  inasmuch  as  these  amino  acids  crystallize  together 
in  such  a  way  that  the  combination  persists  even  after  repeated  recrys- 
tallizations.     Valine  is  dextro-rotatory. 

Arginine,  C6H14N4O2. — Arginine  is  8-guanidine-a-amino-valerianic 
acid  and  possesses  the  following  structural  formula : 

H    H    H    NH2 
NH— C— C— C— C— COOH. 
NH=C        H    H    H    H 

NH2 

It  has  been  obtained  from  every  protein  so  far  subjected  to  decomposi- 
tion. The  arginine  obtained  from  proteins  is  dextro-rotatory,  and  has 
pronounced  basic  properties,  reacts  strongly  alkaline  toHtmus,  and  forms 
stable  carbonates.  Because  of  these  facts,  Kossel  considers  arginine  to 
be  the  nucleus  of  the  protein  molecule.  It  is  obtained  in  widely  different 
amounts  from  different  proteins,  over  85  per  cent  of  certain  protamines 
having  been  obtained  in  the  form  of  this  amino  acid.  It  is  claimed  that 
in  the  ordinary  metabolic  activities  of  the  animal  body  arginine  gives 
rise  to  urea.  \Miile  this  claim  is  probably  true,  it  should,  at  the  same 
time,  be  borne  in  mind  that  the  greater  part  of  the  protein  nitrogen  is 
eliminated  as  urea  and  that,  therefore,  but  a  very  small  part  can  arise 
from  arginine. 

Leucine,  CeHisNOa. — Leucine  is  an  abundant  end-product  of  the 
decomposition  of  protein  material,  and  was  one  of  the  first  of  these 
products  to  be  discovered.  It  is  a-amino-isohulyl-acetic  acid,  and 
therefore  has  the  following  formula: 

CH3      NH2 

I  1 

H— CCH2C— COOH. 

I  1 

CH3     H 

The  leucine  which  results  from  protein  decomposition  is  /-leucine. 
Leucine  is  present  normally  in  the  pancreas,  thymus,  thyroid,  spleen, 
brain,  liver,  kidneys,  and  salivary  glands.  It  has  been  found  patliolog- 
ically  in  the  urine  (in  acute  yellow  atrophy  of  the  liver,  in  acute  phos- 
phorus poisoning,  and  in  severe  cases  of  typhoid  fever  and  smallpox), 
and  in  the  liver,  blood,  and  pus. 

Pure  leucine  crystallizes  in  thin,  white,  hexagonal  plates.     Crystals 


8o 


PHYSIOLOGICAL   CHEMISTRY 


of  pure  leucine  are  reproduced  in  Fig.  28.  It  is  rather  easily  soluble  in 
water  (46  parts),  alkalis,  ammonia,  and  acids.  On  rapid  heating  to 
295°C.,  leucine  decomposes  with  the  formation  of  carbon  dioxide,  ammo- 
nia, and  amylamine.  Aqueous  solutions  of  leucine  obtained  from  pro- 
teins are  levo-rotatory,  but  its  acid  or  alkaline  solutions  are  dextro- 
rotatory. So-called  impure  leucine^  is  a  slightly  refractive  substance, 
which  generally  crystallizes  in  balls  having  a  radial  structure,  or  in 
aggregations  of  spherical  bodies.  Fig.  139,  Chapter  XXIV. 

Isoleucine,  C6H13NO2. — Isoleucine  is  a-amino-^-methyl-^-ethyl-pro- 
■pionic  acid,  and  possesses  the  following  structural  formula* 

CH3     NHo 


H— C 


C— COOH. 


C2H5    H 


Fig.  28. — Leucine. 


This  amino  acid  was  discovered  by  Ehrlich  in  1903.  Its  presence  has 
been  established  among  the  decomposition  products  of  only  a  few  pro- 
teins, although  it  probably  occurs  among  those  of  many  or  most  of  them. 
Ehrlich  has  shown  that  the  J-amyl  alcohol  which  is  produced  by  yeast 
fermentation  originates  from  isoleucine  and  the  isoamylalcohol  origi- 
nates from  leucine.     Isoleucine  is  dextro-rotatory. 

Lysine,  CH2  (NH2)  ■  CH2  •  CH2  •  CH2  •  CH  (NH2)  •  COOH.— The  three 
bodies,  lysine,  arginine,  and  histidine,  are  frequently  classed  together 
as  the  hexone  bases.  Lysine  was  the  first  of  the  bases  discovered.  It 
is  a-e-diammo-caproic  acid  and  hence  possesses  the  following  structure : 

^  These  balls  of  so-called  impure  leucine  do  contain  considerable  leucine,  but  inasmuch 
as  they  may  contain  many  other  things  it  is  a  bad  practice  to  allude  to  them  as  leucine. 


PROTEINS 

NH2H    H    H    XHo 
H— C— C— C— C— C— COOH 
H    H    H    H    H 


81 


Fig.  29. — ^Lysine  Picrate. 

It  is  dextro-rotatory  and  is  found  in  relatively  large  amount  in  casein 
and  gelatin.  Lysine  is  obtained  from  nearly  all  proteins,  but  is  absent 
from  the  vegetable  proteins  which  are  soluble  in  strong  alcohol.     It  is 


Fig.  30. — AsPARTic  Acid. 

the  mother-substance  of  cadaverin  and  has  never  been  obtained  in 
crystalline  form.    Lysine  is  usually  obtained  as  the  picrate  which  is 
sparingly  soluble  in  water  and  crystallizes  readily.     These  crystals  are 
shown  in  Fig.  29. 
6 


82  PHYSIOLOGICAL    CHEMISTRY 

Aspartic  Acid,  C4H7NO4. — Aspartic  acid  is  amino-succinic  acid  and 
has  the  following  structural  formula: 

NH2 
H— CCOOH   . 

H— CCOOH. 

I 
H 

The   amide   of   aspartic   acid,  asparagine,  is  very  widely  distributed 
in   the  vegetable  kingdom.     Asparagine  has   the  following  formula. 

NH2 

H— CCOOH 

I 

H— CC0(NH2). 

H 

The  crystalline  form  of  aspartic  acid  is  exhibited  in  Fig.  30. 

Aspartic  acid  has  been  found  among  the  decomposition  products  of 
all  the  proteins  examined,  except  the  protamines.  It  has  not  been  ob- 
tained, however,  in  very  large  proportion  from  any  of  them.  The 
aspartic  acid  obtained  from  protein  is  levo-rotatory. 

Glutamic  Acid,  C5H9NO4. — This  acid  is  a-amino-normal-glutaric 
acid  and  as  such  bears  the  following  graphic  formula: 

NH2 

I 
H— CCOOH 

H— C— H 

I 
H— CCOOH. 

H 

Glutamic  acid  is  yielded  by  all  the  proteins  thus  far  examined, 
except  the  protamines,  and  by  most  of  these  in  larger  amount  than 
any  other  of  their  decomposition  products.  It  is  yielded  in  especially 
large  proportion  by  most  of  the  proteins  of  seeds,  43.66  per  cent  having 
been  obtained  by  Osborne  and  Guest ^  by  the  hydrolysis  of  gliadin,  the 
prolamin  of  wheat.  This  is  the  largest  amount  of  any  single  decompo- 
sition product  yet  obtained  from  any  protein  except  the  protamines. 

^  Osborne  and  Guest:  Jour.  Biol.  Chcm.   9,  425,  191 1. 


PROTEINS 


83 


Glutamic  acid  and  aspartic  acid  arc  the  only  dibasic  acids  which 
have  thus  far  been  obtained  as  decomposition  products  of  proteins.  As 
there  is  an  apparent  relation  between  the  proportion  of  these  acids 
and  that  of  ammonia  which  the  different  proteins  yield  it  is  possible 
that  one  of  the  carboxyl  groups  of  these  acids  is  united  with  XHo  as 
an  amide,  the  other  carboxyl  group  being  united  in  polypeptide  union 
(see  page  32)  with  some  other  amino  acid.  This  might  be  represented 
by  the  following  formula: 

R— CHNH— COOH 

/ 

CO— CHNHo— CH2— CH2— COXH.2. 

It  has  been  shown  by  Thierfelder  and  Sherwin^  that  the  amide, 
glutamine,  is  a  product  of  normal  metabolism  and  hence  this  substance 
rather  than  glutamic  acid  is  present  in  the  protein  molecule. 


Fig.  31. — Glutamic  Acid. 

Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 

The  glutamic  acid,  yielded  by  proteins  upon  hydrolysis,  is  dextro- 
rotatory.    Crystals  of  glutamic  acid  are  reproduced  in  Fig.  31. 

Proline,  C5H9XO2. — Proline  is  a-pyrro\idinc-carhoxyUc  acid  and 
possesses  the  following  graphic  structure: 

H2C         CH2 


HoC 


CHCnOH. 


NH 


Proline  was  llrst  obtained  as  a  decomposition  product  of  casein.     Pro- 
*  Thierfelder  and  Sherwin:  Zeil.  Physiol.  Chcmic,  94,  1,  1915. 


84 


PHYSIOLOGICAL   CHEMISTRY 


line  obtained  from  proteins  is  levo-rotatory  and  is  the  only  protein  de- 
composition product  which  is  readily  soluble  in  alcohol.  It  is  also  one  of 
the  few  heterocyclic  compounds  obtained  from  proteins.  Proline  has 
been  found  among  the  decomposition  products  of  all  proteins  except  the 


Fig.  32. — LEVo-a-PROLiNE. 

protamines.  The  maximum  yield  reported  is  13.73  P^^  cent  obtained 
by  Osborne  and  Clapp  from  the  hydrolysis  of  hordein.  Fischer  and 
Boehner^  have  obtained  7.7  per  cent  from  the  hydrolysis  of  gelatin. 


Fig.  ^s. — Copper  Salt  of  Proline. 
Reproduced  from  a  micro-photograph  made  by  Prof.  E.  T.  Reichert,  of  the  University  of 

Pennsylvania. 

The  crystalline  form  of  levo-a-proline  is  shown  in  Fig.  32   and  the 
copper  salt  of  prohne  is  represented  by  a  micro-photograph  in  Fig.  7,2). 
The  crystals  of  the  copper  salt  have  a  deep  blue  color,  but  when  they 
^  Fischer  and  Boehner:  Zeit.  phys.  chem.,  65,  118,  1910. 


PROTEINS  85 

lose  their  water  of  crystallization  they  assume  a  characteristic  violet 
color. 

Oxyproline,  C5H9NO3. — Oxyproline  was  discovered  by  Fischer. 
It  has  as  yet  been  obtained  from  only  a  few  proteins,  but  this  may  be 
due  to  the  fact  that  only  a  few  have  been  examined  for  its  presence. 
The  position  of  the  hydroxyl  group  has  not  yet  been  established. 

Diaminotrihydroxydodecanoic  Acid,  C12H26N2O6. — This  amino  acid 
was  discovered  by  Fischer  and  Abderhalden  as  a  product  of  the  hydro- 
lysis of  casein.  It  has  thus  far  been  obtained  from  no  other  source. 
It  is  levo-rotatory  and  its  constitution  has  not  been  determined. 

Experiments 

Protein  Decomposition. — While  the  ordinary  courses  in  physiological 
chemistry  preclude  any  extended  study  of  the  decomposition  products 
of  proteins,  the  manipulation  of  a  simple  decomposition  and  the  sub- 
sequent isolation  and  study  of  a  few  of  the  products  most  easily  and 
quickly  obtained  will  not  be  without  interest.^  To  this  end  the  student 
may  use  the  following  decomposition  procedure. 

Treat  the  protein  (coagulated  egg  albumin)  in  a  large  flask  with  water  con- 
taining 3-5  per  cent  of  H2SO4  and  place  it  on  a  water-bath  until  the  protein  ma- 
terial has  been  decomposed  and  there  remains  a  fine,  fluffy,  insoluble  residue. 
Filter  off  this  residue  and  neutralize  the  filtrate  with  Ba(0H)2  and  BaCOs* 
Filter  off  the  precipitate  of  BaS04  which  forms  and  when  certain  that  the  fluid  is 
neutral  or  faintly  acid,-  concentrate  (first  on  a  wire  gauze  and  later  on  a  water- 
bath)  to  a  syrup.  This  syrup  contains  the  end-products  of  the  decomposition  of 
the  protein,  among  which  are  proteoses,  peptones,  tyrosine,  leucine,  etc.  Add 
95  per  cent  alcohol  slowly  to  the  warm  syrup  until  no  more  precipitate  forms, 
stirring  continuously  with  a  glass  rod.  This  precipitate  consists  of  proteoses  and 
peptones.  Gather  the  sticky  precipitate  on  the  rod  or  the  sides  of  the  dish  and, 
after  warming  the  solution  gently  for  a  few  moments,  filter  it  through  a  filter 
paper  which  has  not  been  previously  moistened.  After  dissolving  the  precipi- 
tate of  proteoses  and  peptones  in  water^  the  solution  may  be  treated  according  to 
the  method  of  separation  given  on  page  120. 

The  leucine  and  tyrosine,  etc.,  are  in  solution  in  the  warm  alcoholic  filtrate. 
Concentrate  this  filtrate  on  the  water-bath  to  a  thin  syrup,  transfer  it  to  a  beaker, 
and  allow  it  to  stand  over  night  in  a  cool  place  for  crystaUization.  The  tyrosine 
first  crystallizes  (Fig.  25,  page  76),  followed  later  by  the  formation  of  characteristic 

1  The  procedure  here  set  forth  has  nothing  in  common  with  the  procedure  by  means 
of  which  the  long  line  of  decomposition  products  just  enumerated  are  obtained.  This 
latter  process  is  an  exceedingly  comphcated  one  which  is  entirely  outside  the  province  of 
any  course  in  physiological  chemistry. 

^  If  the  solution  is  alkaline  in  reaction  at  this  point,  the  amino  acids  will  be  broken 
down  and  ammonia  will  be  evolved. 

'  At  this  point  the  aqueous  solution  of  the  proteoses  and  peptones  may  be  filtered  to 
remove  any  BaS04  which  may  still  remain.  Tyrosine  crystals  will  also  be  found  here, 
since  it  is  less  soluble  than  the  leucine  and  may  adhere  to  the  proteose-peptone  precipitate. 
Add  the  crystals  of  tyrosine  to  the  warm  alcohol  filtrate. 


86  PHYSIOLOGICAL    CHEMISTRY 

crystals  of  impure  leucine  (see  Fig.  139,  Chapter  XXIV).  After  examining  these 
crystals  under  the  microscope,  strain  ofif  the  crystalline  material  through  fine 
muslin,  heat  it  gently  in  a  little  water  to  dissolve  the  leucine  (the  tyrosine  will  be 
practically  insoluble)  and  filter.  Concentrate  the  filtrate  and  allow  it  to  stand  in  a 
cool  place  over  night  for  the  crude  leucine  to  crystallize.  Filter  off  the  crystals 
and  use  them  in  the  tests  for  leucine  given  on  page  87.  The  crystals  of  tyrosine 
remaining  on  the  paper  from  the  first  filtration  may  be  used  in  the  tests  for  tyro- 
sine as  given  below.  If  desired,  the  tyrosine  and  leucine  may  be  purified  by 
recrystallizing  in  the  usual  manner.  Habermann  has  suggested  a  method  of 
separating  leucine  and  tyrosine  by  means  of  glacial  acetic  acid. 

Experiments  on  Tyrosine 

Make  the  following  tests  with  the  tyrosine  crystals  prepared  in 
the  above  experiments,  or  upon  those  obtained  during  the  preparation 
of  cystine  (see  page  87),  or  upon  some  pure  tyrosine  furnished  by  the 
instructor. 

1.  Microscopical  Examination. — Place  a  minute  crystal  of  tyrosine  on  a  slide, 
add  a  drop  of  water,  cover  with  a  cover-glass,  and  examine  microscopically. 
Now  run  more  water  under  the  cover-glass  and  warm  in  a  Bunsen  flame  until  the 
tyrosine  has  dissolved.  Allow  the  solution  to  cool  slowly,  then  examine  again 
microscopically,  and  compare  the  crystals  with  those  shown  in  Fig.  25,  page  76. 

2.  Solubility. — Try  the  solubility  of  very  small  amounts  of  tyrosine  in  cold  and 
hot  water,  cold  and  hot  95  per  cent  alcohol,  dilute  NH4OH,  dilute  KOH  and  dilute 
HCl. 

3.  Sublimation. — Place  a  little  tyrosine  in  a  dry  test-tube,  heat  gently  and 
notice  that  the  material  does  not  sublime.  How  does  this  compare  with  the 
result  of  Experiment  3  under  Leucine? 

4.  Hoffman's  Reaction. — This  is  the  name  given  to  Millon's  reaction  when 
employed  to  detect  tyrosine.  Add  about  3  c.c.  of  water  and  a  few  drops  of  Mil- 
lon's reagent  to  a  little  tyrosine  in  a  test-tube.  Upon  dissolving  the  tyrosine  by 
heat  the  solution  gradually  darkens  and  may  assume  a  dark  red  color.  What 
group  does  this  test  show  to  be  present  in  tyrosine? 

5.  Sulphuric  Acid  Test  (Piria). — Warm  a  little  tyrosine  on  a  watch  glass  on  a 
boiling  water-bath  for  20  minutes  with  3-5  drops  of  cone.  H2SO4.  Tyrosine- 
sulphuric  acid  is  formed  in  the  process.  Cool  the  solution  and  wash  it  into  a 
small  beaker  with  water.  Now  add  CaCOs  in  substance  slowly  with  stirring, 
until  the  reaction  of  the  solution  is  no  longer  acid.  Filter,  concentrate  the 
filtrate,  and  add  it  to  a  few  drops  (avoid  an  excess)  of  very  dilute  neutral  ferric 
chloride.  A  purple  or  violet  color,  due  to  the  formation  of  the  ferric  salt  of 
tyrosine-sulphuric  acid,  is  produced.  This  is  one  of  the  most  satisfactory  tests 
for  the  identification  of  tyrosine. 

6.  Formaldehyde -Sulphuric  Acid  Test  (Momer). — ^Add  about  3  c.c.  of 
M timer's  reagent'  to  a  little  tyrosine  in  a  test-tube,  and  gently  raise  the  tempera- 
ture to  the  boiling-point.     A  green  color  results. 

^  Morner's  reagent  is  prepared  by  thoroughly  mixing  i  volume  of  formalin,  45  volumes 
of  distilled  water,  and  55  volumes  of  concentrated  sulphuric  acid. 


PROTEINS  87 

7.  Folin  and  Denis's  Test.'^ — To  12  c.c.  of  the  solution  to  be  tested  add  an 
equal  volume  of  a  special  reagent  (containing  10  per  cent  sodium  tungstate,  2 
per  cent  phosphomolybdic  acid  and  10  per  cent  phosphoric  acid)  and  3-10  c.c. 
of  a  saturated  solution  of  sodium  carbonate.  A  blue  color  indicates  tyrosine. 
It  is  said  to  detect  i  part  in  one  million. 

Abderhalden-  claims  the  reagent  also  reacts  with  tryptophane, 
oxytryptophane  and  /-oxyproline. 

Experiments  on  Leucine 

Make  the  following  tests  upon  the  leucine  crystals  already  prepared 
or  upon  some  pure  leucine  furnished  by  the  instructor. 

I,  2  and  3.  Repeat  these  experiments  according  to  the  directions  given 
under  Tyrosine  (pages  86  and  87 j. 

Preparation  of  Cystine^ 

From  50  to  500  grams  of  wool  or  hair  is  pushed  into  a  (Jena)  flask  and  con- 
centrated hydrochloric  acid  (200  c.c.  to  each  100  grams  of  wool)  is  added.  In 
order  to  get  a  part  of  the  acid  quickly  to  the  bottom  of  the  flask  a  part  of  the  acid 
may  be  put  in  first,  then  the  wool,  and  finally  the  remaining  acid.  A  condenser 
consisting  only  of  a  glass  tube  2  to  3  ft.  long  is  inserted  and  the  mixture  is  boiled 
until  the  biuret  reaction  is  entirely  negative.  The  wool  dissolves  in  a  few  min- 
utes and  if  much  cystine  is  desired  more  wool  and  acid  can  then  be  introduced. 
After  three  to  five  hours'  boiling  with  moderate  quantities  of  wool  the  biuret 
reaction  has  usually  disappeared. 

To  the  hot  acid  solution  of  amino  acids  so  obtained  is  added  at  once  an  excess 
of  solid  sodium  acetate,  i.e.,  until  the  Congo  red  reaction  for  mineral  acids  is 
entirely  negative.  A  dark,  heavy  precipitate  containing  practically  all  the  cystine 
is  obtained.  After  a  few  hours'  standing  at  room  temperature  the  liquid  is  filtered 
off  and  the  precipitate  is  washed  with  cold  water.  (From  the  mother  liquor 
diluted  with  the  wash  water  is  usually  obtained  on  long  standing  a  second  pre- 
cipitate consisting  chiefly  of  tyrosine.) 

The  crude  cystine  is  then  dissolved  in  boiUng  3-5  per  cent  hydrochloric  acid 
and  the  solution  is  decolorized  with  good  boneblack  which  should  have  been 
previously  thoroughly  digested  with  hot,  dilute  hydrochloric  acid  and  then  washed 
with  water  in  order  to  remove  the  calcium  phosphate.  The  hot  filtrate  from  the 
boneblack  should  be  as  clear  as  water.  If  it  is  not  perfectly  colorless  the  bone- 
black  treatment  should  be  repeated  and  if  a  colorless  solution  is  not  then  ob- 
tained the  fault  lies  with  the  quaUty  of  the  boneblack.  The  last  filtrate  is  heated 
to  boiUng  and  the  cystine  precipitated  by  a  slow  addition  of  concentrated  hot 
sodium  acetate  solution. 

Large  amounts  of  colorless  cystine  consisting  of  tjrpical  hexagonal  plates  can 
thus  be  prepared  without  difficulty  and  with  very  little  labor.  Compare  the 
microscopical  appearance  of  these  crystals  with  those  shown  in  Fig.  26,  page  76. 

'  Folin  and  Denis:  Jour.  Biol.  C/icm.,  12,  245,  IQ12. 
*  Abderhalden:  Zeit.  Physiol.  CItem.,  85,  91,  1913. 
'  Folin:  Jour.  Biol.  Cliem.,  8,  9,  1910. 


PHYSIOLOGICAL   CHEMISTRY 


THE  QUANTITATIVE  DETERMINATION  OF  ALIPHATIC 
AMINO  GROUPS 

Method  of  Van  Slyke.^ — Principle. — This  method  for  the  determination  of 
ahphatic  amino  nitrogen  is  based  on  the  measurement  of  the  nitrogen  gas  evolved 
in  the  reaction, 

RNH2+HN02  =  ROH+N2+HoO. 

During  the  process  the  following  reaction  also  takes  place,  the  nitrous  acid  solution 
decomposing  spontaneously  with  the  formation  of  nitric  oxide, 

2HN02  =  HN03+NO. 


Fig.  34. 


-Van  Slyke  Amino  Nitrogen 
Apparatus. 


Fig.  35. — Section  of  Van  Slyke 
Apparatus. 


This  latter  reaction  is  utiUzed  in  displacing  all  the  air  of  the  apparatus  with  nitric 
oxide.  The  amino  solution  is  then  introduced,  evolution  of  nitrogen  mixed  with 
nitric  oxide  resulting.  The  oxide  is  absorbed  with  alkaline  permanganate  solution 
and  the  pure  nitrogen  measured  in  a  special  gas  burette  shown  in  the  figure. 
Procedure. — The  determination  is  carried  out  in  three  stages: 
I.  Displacement  of  Air  by  Nitric  Oxide. — Water  f rom  F  (see  Figs.  34  and  35), 
fills  the  capillary  leading  to  the  Hempel  pipette  and  also  the  other  capillary 
as  far  as  c.     Into  A  one  pours  a  volume  of  glacial  acetic  acid  sufficient  to  fill 

^  Van  Slyke:  Jour.  Biol.  Chem.,  12,  275,  1912;  61,  121  and  125,  1913. 


PROTEINS  89 

one-fifth  of  D.  For  convenience,  A  is  etched  with  a  mark  to  measure  this  amount. . 
The  acid  is  run  into  D  cock  c  being  turned  so  as  to  let  the  air  escape  from  D. 
Through  A  one  now  pours  sodium  nitrite  solution  (30  grams  NaNOa  to  100  c.c. 
H2O)  until  D  is  full  of  solution  and  enough  excess  is  present  to  rise  a  little  above 
the  cock  into  A.  It  is  convenient  to  mark  A  for  measuring  off  this  amount  also. 
The  gas  exit  from  D  is  now  closed  at  c,  and,  a  being  open,  D  is  shaken  for  a  few 
seconds.  The  nitric  oxide,  which  instantly  collects,  is  let  out  at  c,  and  the  shaking 
repeated.  The  second  crop  of  nitric  oxide  which  washes  out  the  last  portions  of  air, 
is  also  let  out  at  c.  D  is  now  connected  with  the  motor  and  shaken  till  all  but  20  c.c. 
of  the  solution  have  been  displaced  by  nitric  oxide  and  driven  back  into  A .  A  naark 
on  D  indicates  the  20  c.c.  point.  One  then  closes  a  and  turns  c  and /so  that  D  and 
F  are  connected.     The  above  manipulations  require  between  one  and  two  minutes. 

2.  Decomposition  of  the  Amino  Substance. — Of  the  amino  solution  to  be  analyzed 
10  c.c.  or  less,  as  the  case  may  be,  are  measured  off  in  B.  Any  excess  added  above 
the  mark  can  be  run  off  through  the  outflow  tube.  The  desired  amount  is  then  run 
into  D,  which  is  already  connected  with  the  motor,  as  shown  in  Fig.  34.  It 
is  shaken  when  a-amino  acids  are  being  analyzed  for  a  period  of  three  to  five 
minutes.  With  a-amino  acids,  proteins  or  partially  or  completely  hydrolyzed 
proteins,  we  find  that  at  the  most  five  minutes  vigorous  shaking  completes  the 
reaction.  Only  in  the  case  of  some  native  proteins  which,  when  deaminized  form 
unwieldy  coagula  that  mechanically  interfere  with  the  thorough  agitation  of  the 
mixture,  a  longer  time  may  be  required.  In  case  a  viscous  solution  is  being  analyzed 
and  the  liquid  threatens  to  foam  over  into  F,  B  is  rinsed  out  and  a  little  caprylic 
alcohol  is  added  through  it.  For  amino  substances  such  as  amino  purins,  requiring 
a  longer  time  than  five  minutes  to  react,  one  merely  mixes  the  reacting  solutions 
and  lets  them  stand  the  required  length  of  time,  then  shakes  about  two  minutes  to 
drive  the  nitrogen  completely  out  of  solution. 

When  it  is  known  that  the  solution  to  be  analyzed  is  likely  to  foam  violently, 
it  is  advisable  to  add  caprylic  alcohol  through  B  before  the  amino  solution.  B  is 
then  rinsed  with  alcohol  and  dried  with  ether  or  a  roll  of  filter  paper  before  it 
receives  the  amino  solution. 

3.  Absorption  of  Nitric  Oxide  and  Measurement  of  Nitrogen. — The  reaction  being 
completed,  all  the  gas  in  D  is  displaced  into  F  by  liquid  from  A  and  the  mixture  of 
nitrogen  and  nitric  oxide  is  driven  from  F  into  the  absorption  pipette.^  The  driving 
rod  is  then  connected  with  the  pipette  by  lifting  the  hook  from  the  shoulder  of  d  and 
placing  the  other  hook,  on  the  opposite  side  of  the  driving  rod,  over  the  horizontal 
lower  tube  of  the  pipette.  The  latter  is  then  shaken  by  the  motor  for  a  minute, 
which,  with  any  but  almost  completely  exhausted  permanganate  solutions,  com- 
pletes the  absorption  of  nitric  oxide.  The  pure  nitrogen  is  then  measured  in  F. 
During  the  above  operations  a  is  left  open,  to  permit  displacement  of  liquid  from  D 
as  nitric  oxide  forms  in  D. 

Blank  determinations,  performed  as  above  except  that  10  c.c.  of  distilled  water 
replaces  the  solution  of  amino  substance,  must  be  performed  on  every  fresh  lot 
of  nitrite  used.  Nitrite  giving  a  much  larger  correction  than  0.3  to  0.4  c.c.  should 
be  rejected. 

The  room  temperature  and  the  barometric  pressure  must  be  noted.     The 

*The  solution  in  the  absorption  pipette  is  40  grams  KMNO4  and  25  grams  KOH  in  a 
liter. 


90 


PHYSIOLOGICAL    CHEMISTRY 


MILLIGRAMS  OF  AMINO  NITROGEN  CORRESPONDING  TO  i  C.C.  OF  NITRO- 
GEN GAS  AT  ii°-3o°C.;  728-772  MM.  PRESSURE 


13° 
14° 
15° 


728 


732 


734 


736    I     738 


0.s68o|o.s69S  O.SSio  0.5725 

0.565s  0.56700.5685  0.S700 

o".  5630  o .  5645  o .  5660  o.  567s 

0.5605  0.56200.5635  0.5650I0. 5665  0.5680 

0.55800.5595,0.5610  0.5625^0.  s640|0. 5655 


0.574SI0.5760I0.5775 

0.5720  0.5735  0.5750 

0.5695  0.57100.5725 

0.5700 

0.5670 


0.S790 
0.5765 
0.5740 


744 


0.5805 
0.5780 
0.5755 


0.5715  0.5730 


746 


748 


750 


0.5685  0.570s  0.5720b. 5735 !o.S7SO 


0.5820  0.5840  0.5855 
0.57950.58150.5830 
0.5770|o.5785|o.5805 
0.5745:0.5760,  .05775 


13" 
14° 
is" 


16°  0.555s  0.55700.5585  0.56000.5615  0.5630 
17°  0.552s|o. 55400. 5555  0.5575J0. 55900. 560s 
18°  0.55000.5515  0.55300.55450. 5560  0.5580 
19°  [0.5475:0.5490  0.5505  6.5520  0.5535  0.5550 
o.  5445  0.54600.5475  0.54950. 5510  0.5525 


0.5645 
0.5620 
0.5595 


0.5660  0.5675 
0.5635:0.5650 
0.56100. 562s 


0.5690b. 571010.5725 
o . 566s  o . 56800 . 5695 
0.5640  0.5655  0.5670 


0.5565  0.5580b.  5595  0.5610  o.5630jO.  5645 
0.5540  0.5555  0.55700.5585  0.56000.5615 


16" 
17° 
18° 
19° 


23° 
24° 
25° 


0.54200.5435 
0.53950.5410 


0.54500.5465  0.54800.5495  0.55100.5525  0.5540 
0. 542510. 5440j0. 5455  0.5470  0.548s  0.5500  o.SSiS 
0. 5365^0. 5380b.  53950.  5410,0.5425  0.5440 
0.5335  o.S350|0.  5365  0.53800.54000.541510.543010.5445  0.5460 
0.5310I0.5325  0.5340  0.5355,0.53700.5385  0.5400  0.5415  0.5430 


0.555s  0.5575JO. 5590 
o. 5530,0. 554SJO. 5560 
0.5500b. 5515  0.5530 
o.  5475  ;o.5490|0. 550s 
0.5445:0.54600.5475 


23 

24° 

25° 


26" 

27" 

28° 

29° 

130° 


0.52600.5295  0.53100.5325  0.53400.5355 
0.5250b. 5265(0. 5280  0.5295  0.53100.532s 
0.5220  0.523s  o.  5250  0.5265I0. 5280:0. 5295 
0.5195  o.  52 lojo.  5220  0.523510.52500.5265 
0.5160  0.517s  0.51900.520510.522010.5235 


0.5370 
0.5340 
0.5310 
0.5280 


0.5365  0.5400 
0.5355b. 5370 


0.5325  0.5340  0.535s  0.5370 
0.5295  0.5310  0.53250. 5340 


0.54IS  0.5430  0.544s 
0.538510.54000.5415 
0.5385 

0.5355 


0.52500.5265  0.5280  0.5295  0.5310 


0.532s 


260 

2  7" 

28° 
29" 
30° 


t 


728 


730 


732 


734         736     I   738 


740 


742 


746 


748 


754    I    7S6 


7S8 


.5870 
■  584s 
.5820 
.5790 
.5765 


o.  5885  |o.  59000. 

o. 5860b. 5875  o, 

o.s835j0.58sojo, 

0.580S  0.582s  o, 

576510. 57950, 


760 


5915  o. 
5890  o. 

58650. 
5840:0. 
581010. 


762 


764   766   768 


770 


5935  O.595O1O 
S90S  0.5925  O 
58800.5895:0 


S8S5 
S830 


o.5870|0 
0.58450 


59650. 
59400. 
5910  o. 
5885'o. 
,5860  o. 


59800. 
S9S5|o, 
S930JO. 
59000, 
58750. 


599Sb 
597o!o 
594sjo 
59150 
5890I0 


6010 

5985 
5960 
5935 
5905 


0.6030 
O . 6000 
0.597S 
O.S9SO 
0.5920 


772 


13" 
14° 
IS" 


5740 
S7IO 
568s 
5660 
5630 


0.57S5b.S770JO 
57300.5745  O 
o. 5700b. 5715  O 
0.567s  0.5690  O 
0.5645  0.5660  o 


5785  o. 
576o'o. 
5730|0. 
S705:o, 
.56750, 


I  i 

5800b. 58150. 

S77SjO.S790  0. 
S74s|o.576sjo. 
5720|0.5735  O. 
569O1O.570S  O. 


58300. 
5805  O, 
5780  O. 
5750  O. 
5725  O. 


58500. 
5820  O. 
579510. 
57650. 
574oio. 


I 
5865JO. 
S82s'o. 
5810J0. 
5780  O. 
575510. 


5880 
5850 
582s 
5795 
5770 


0.589s 
0.586s 
0.5840 
0.5810 
0.578s 


16° 
17° 
18° 
19° 


23° 
24° 
25° 


0.560s 
O.SS7S 
O.S545 
0.5520 
0.S490 


0.5620  0.5635  0.56so]o.s665  0.5680,0.5695  0.57IOJO.  5725 |o.  5740 
o.5S90io. 5605  0.5620  0.5635  0.5650  0.5665  0.5680(0.5695  0.5715 


0.55600.5575  0.5595 
0.5535  0.55500.5565 
0.5505  0.5520  O.SS3S 


0.5610b. 5625  0.5640  0.56ssb.567ob.5685 
0.55800.5595  0.56100.5625  0.56400.5655 
0.55500.5565  0.558010. 5595(0. 5610  0.5625 


0.S7SS 
0.5730 
0.5700 
0.5670 
o . 5640 


23° 
24° 
2S° 


26° 
27° 
28° 
29° 
30» 


0.5460 
0.5430 
0.5400 
0.5370 
0.5340 


0.5475:0.54900.5505  0.55200.5535  0.5550(0.5565  0.55800.5595 
0. 544Sb.5460|0. 5475 jo.  5490J0.  5505  0.5520  0.5535(0. 555o|o.  5565 
0.5415  0.54300.5445,0.5460,0.5475  0.5490, 0.5505  0.5520 


0.538510.5400 
0.53550.5370 


0.5415 
5385 


0.543o|o.  5445,0.5460  0.5475  0.5490 
0.5400  0.541 5  0.5430  0. 5445 '0.5460 


5535 
0.550s 
5475 


0.5610 
0.5580 
0.5550 
0.5520 
0.5490 


26° 
27° 
28° 
29° 
30° 


752 


754 


756 


7S8 


760 


762    I    764 


766    I    768 


770 


772 


Journal  of  Biological  Chemistry, 
mination  of  Amino  Groups. 


12,  27s,  1Q12.     Van  Slyke:  The  Quantitative  Deter- 


PROTEINS  91 

calculation  of  the  weight  of  nitrogen  gas  corresponding  to  the  volume  obtained  is 
most  readily  made  with  the  aid  of  the  tables  (see  page  90)  devised  for  this  purpose.^ 

The  Van  Slyke  Micro-apparatus.  ^ — In  later  work.  \an  Slyke  has  used  to  a  large 
extent  an  ai)paratus  which  differs  from  the  one  described  above  only  in  being  con- 
siderabl}-  smaller.  More  accurate  measurements  can  be  made  with  this  and 
smaller  amounts  of  amino  nitrogen  determined.  In  using  this  only  10  c.c.  of 
nitrite  solution  and  2.5  c.c.  of  acetic  acid  are  required  for  an  analysis.  One-fifth 
the  amount  of  substance  may  be  analyzed  with  the  same  degree  of  accuracy  as 
with  the  larger  apparatus.  Practically  the  only  alteration  from  the  mode  of  opera- 
tion already  detailed  above,  is  in  the  speeds  at  which  the  deaminizing  bulb  and  the 
Hempel  pipette  are  shaken.  During  the  first  stage  of  the  analysis  the  deaminizing 
bulb  should  be  shaken  by  the  motor  at  a  very  high  rate  of  speed,  about  as  fast  as 
the  eye  can  follow  or  an  unnecessary  amount  of  time  is  lost  in  freeing  the  apparatus 
from  air.  This  stage  is  also  much  accelerated  by  warming  the  nitrite  solution  to 
30°  before  it  is  used,  in  case  a  low  room  temperature  has  reduced  the  temperature 
of  the  solutions  below  20°.  In  the  third  stage  when  the  nitric  oxide  is  being  ab- 
sorbed by  the  permanganate,  the  Hempel  pipette  should  be  shaken  not  faster  than 
twice  per  second.     This  is  to  prevent  the  breaking  off  of  small  gas  bubbles. 

It  is  especially  necessary  that  in  the  first  stage  the  removal  of  air  be  complete. 
This  is  assured  by  shaking  the  solution  in  the  deaminizing  bulb  back  each  time, 
in  this  stage,  until  the  bulb  is  two-thirds  filled  w-ith  nitric  oxide. 

For  the  determination  of  total  and  free  amino  acid  nitrogen  in  the  urine  by 
this  method  see  chapter  on  Quantitative  Analysis  of  Urine. 

ESTIMATION  OF  AMINO -ACED  ^-NITROGEN 

Method  of  Harding  and  MacLean.' — Principle. — Amino-acid  mixtures  when 
treated  with  triketohydrindene  hydrate  give  a  colored  solution  which  may  be  com- 
pared colorimetrically  with  a  standard. 

Procedure. — One  c.c.  of  the  solution  to  be  estimated  (containing  not  more  than 
0.05  mg.  of  amino-acid  a- nitrogen  and  neutral  to  phenolphthalein,  is  mixed  with 
I  c.c.  of  a  ID  per  cent  aqueous  solution  of  pure  pyridine  and  i  c.c.  of  a  freshly  pre- 
pared 2  per  cent  solution  of  triketohydrindene  hydrate  and  heated  in  a  rapidly 
boiling  constant-level  water-bath  for  20  minutes.  At  the  end  of  that  time  the  test 
tube  is  removed,  cooled  and  diluted  to  a  suitable  volume,  usually  100  c.c,  but  if  the 
amino-acid  a-nitrogen  is  very  small  in  amount  a  correspondingly  smaller  dilution 
can  be  used.  The  solution  is  compared  with  a  standard  in  a  Duboscq  colorimeter. 
The  standard  solution  is  prepared  by  dissolving  0.3178  gm.  of  pure,  freshly  crystal- 
lized alanine  in  a  liter  of  distilled  water.  The  solution  contains  0.05  mg.  of  N  per 
c.c.  Treat  i  c.c.  of  this  standard  just  as  above,  except  that  only  i  c.c.  of  trike- 
tohydrindene is  required.  The  standard  solution  is  stable  for  three  months. 
Amounts  of  amino  nitrogen  from  0.005  to  0.05  mg.  may  be  determined.  The 
method  is  inaccurate  for  cystine  and  has  not  yet  been  adapted  for  use  with  biolog- 
ical fluids  other  than  solutions  of  protein  hydrolysis  products. 

1  See  \'an  Slyke:  Jour.  Biol.  Client..  12,  275,  191 2  or  Gatterniann:  Pra.\is  dcs  organis- 
clien  Cliritiikir.K.  ninth  edition.  In  using  the  tables  in  the  latter  work  or  similar  tables  it 
should  be  borne  in  mind  that  the  volume  of  nitrogen  gas  must  be  divided  by  two,  inasmuch 
as  only  one-half  of  the  nitrogen  collected  comes  from  the  amino  groups. 

°  Either  of  these  apparatus  may  be  obtained  from  Kmil  (ireiner,  45  Cliff  Street,  New 
York,  or  from  Robert  Goetze,  Leipzig.  Van  Slyke  has  recently  described  a  third  form  of 
his  apparatus  about  half  of  the  size  of  the  earlier  micro-apparatus.  This  has  a  more 
accurate  burette  so  that  the  gas  volumes  can  be  read  to  0.00 1  c.c.  (,\'an  Slyke:  Jot4r.  Biol. 
Cheni.,  2;^,  407,  1915.) 

^Harding  and  ^lacLean:  Jour.  Biol.  Chem.,  20,  217,  1915. 


CHAPTER  V 

PROTEINS:  THEIR  CLASSIFICATION  AND 
PROPERTIES 

From  what  has  already  been  said  in  Chapter  IV  regarding  the 
protein  substances  it  will  be  recognized  that  the  grouping  of  the  diverse 
forms  of  this  class  of  substances  in  a  logical  manner  is  not  an  easy 
task.  The  fats  and  carbohydrates  may  be  classified  upon  the  funda- 
mental principles  of  their  stereo-chemical  relationships,  whereas  such  a 
system  of  classification  in  the  case  of  the  proteins  is  absolutely  im- 
possible since,  as  we  have  already  stated,  the  molecular  structure  of 
these  complex  substances  is  unknown.  Because  of  the  diversity  of 
standpoint  from  which  the  proteins  may  be  viewed,  relative  to  their 
grouping  in  the  form  of  a  logically  classified  series,  it  is  obvious  that 
there  is  an  opportunity  for  the  presentation  of  classifications  of  a  widely 
divergent  character.  The  fact  that  there  were  until  recent  years  at 
least  a  dozen  different  classifications  which  were  recognized  by  various 
groups  of  English-speaking  investigators  emphasizes  the  dififlculties  in 
the  way  of  the  individual  or  individuals  who  would  offer  a  classification 
which  should  merit  universal  adoption.  ReaHzing  the  great  handi- 
cap and  disadvantage  which  the  great  diversity  of  the  protein  classifi- 
cations was  forcing  upon  the  workers  in  this  field,  the  Chemical  and 
Physiological  Societies  of  England  drafted  a  classification  which  ap- 
pealed to  these  groups  of  scientists  as  fulfilling  all  requirements  and 
presented  it  for  the  consideration  of  the  American  Physiological  Society 
and  the  American  Society  of  Biological  Chemists.  The  outcome  of 
this  has  been  that  there  are  now  only  two  protein  classifications  which 
are  recognized  by  Enghsh-speaking  scientists,  one  the  British  Classi- 
fication, the  other  the  American  Classification.  These  classifications 
are  very  similar  and  doubtless  will  ultimately  be  merged  into  a  single 
classification.  In  our  consideration  of  the  proteins  we  shall  conform 
in  all  details  to  the  American  Classification.  In  this  connection  we 
will  say,  however,  that  we  fell  that  the  English  Societies  have  strong 
grounds  for  preferring  the  use  of  the  term  scleroproteins  for  albu- 
minoids and  chromoproteins  for  hemoglobins.  The  two  classifications 
are  as  follows: 

92 


PROTEINS  93 

CLASSIFICATION  OF  PROTEINS  ADOPTED  BY  THE  AMERI- 
CAN PHYSIOLOGICAL  SOCIETY  AND  THE  AMERICAN 
SOCIETY  OF  BIOLOGICAL  CHEMISTS 

I.  SIMPLE  PROTEINS 

Protein  substances  which  yield  only  a-amino  acids  or  their  deriva- 
tives on  hydrolysis. 

{a)  Albumins. — Soluble  in  pure  water  and  coagulable  by  heat, 
e.g.,  ovalbumin  from  egg  white,  serum  albumin  from  blood  serum, 
lactalbumin  from  milk,  vegetable  albumins. 

(b)  Globulins. — Insoluble  in  pure  water  but  soluble  in  neutral 
solutions  of  salts  of  strong  bases  with  strong  acids, ^  e.g.,  serum  globulin, 
ovoglobulin  from  egg  yolk,  edestin  from  hemp  seed,  amandin  from  almond 
and  peach  kernel,  and  other  vegetable  globulins. 

(c)  Glutelins. — Simple  proteins  insoluble  in  all  neutral  solvents,  but 
readily  soluble  in  very  dilute  acids  and  alkalis,^  ^.g.,  glutenin  from  wheat. 

(d)  Alcohol-soluble  Proteins  (Prolamins).^ — Simple  proteins  sol- 
uble in  70-So  per  cent  alcohol,  insoluble  in  water,  absolute  alcohol,  and 
other  neutral  solvents,"^  e.g.,  zein  from  corn,  gliadin  from  wheat  and 
rye,  hordein  from  barley,  and  bynin  from  malt. 

(e)  Albiuninoids.— Simple  proteins  possessing  a  similar  structure  to 
those  already  mentioned,  but  characterized  by  a  pronounced  insolubihty 
in  all  neutral  solvents,^  e.g.,  elastin  from  ligament,  collagen  from  tendon, 
keratin  from  horn  and  hoof. 

(/)  Histones. — Soluble  in  water  and  insoluble  in  very  dilute  ammor 
nia,  and,  in  the  absence  of  ammonium  salts,  insoluble  even  in  excess  of 
ammonia;  yield  precipitates  with  solutions  of  other  proteins  and  acoagu- 
lum  on  heating  which  is  easily  soluble  in  very  dilute  acids.  On  hydroly- 
sis they  yield  a  large  number  of  amino  acids  among  which  the  basic 
ones  predominate.  In  short,  histones  are  basic  proteins  which  stand 
between  protamines  and  true  proteins,  e.g.,  globin  from  hemoglobin, 
scombronc  from  mackerel  sperm,  thymus  Jrislonc. 

ig)  Protamines.— Simpler  polypeptides  than  the  proteins  included 
in  the  preceding  groups.     They  are  soluble  in  water,  uncoagulable  by 

^  The  precipitation  limits  with  ammonium  sulphate  should  not  be  made  a  basis  for  dis- 
tinguishing the  albumins  from  the  globulins. 

2  Such  substances  occur  in  abundance  in  the  seeds  of  cereals  and  doubtless  represent  a 
weU-defined  natural  group  of  simple  proteins. 

'  The  name  proliimins  has  been  suggested  for  these  alcohol-soluble  proteins  by  Dr. 
Thomas  B.  Osborne  {Science,  190S,  xxviii,  p.  417).  It  is  a  very  fitting  term  inasmuch  as 
upon  hydrolysis  they  yield  particularly  large  amounts  of  proline  and  ammonia. 

*  The  subclasses  defined  (a,  b,  c,  d,)  are  exemplified  by  proteins  obtained  from  both 
plants  and  animals.  The  use  of  appropriate  prefixes  will  sufiice  to  indicate  the  origin  of 
the  compounds,  e.g.,  owglobulin,  /jiValbuniin,  etc. 

*  These  form  the  principal  organic  constituents  of  the  skeletal  structure  of  animals  and 
also  their  external  covering  and  its  appendages.  This  definition  does  not  provide  for 
gelatin  which  is,  however,  an  artificial  derivative  of  collagen. 


94  PHYSIOLOGICAL    CHEMISTRY 

heat,  have  the  property  of  precipitating  aqueous  solutions  of  other  pro- 
teins, possess  strong  basic  properties  and  form  stable  salts  with  strong 
mineral  acids.  They  yield  comparatively  few  amino  acids,  among 
which  the  basic  ones  predominate.  They  are  the  simplest  natural 
proteins,  e.g.,  salmine  from  salmon  sperm,  sturine  from  sturgeon  sperm, 
cln peine  irom.  herring  s per 711,  scombrine  from  mackerel  sperm. 

II.  CONJUGATED  PROTEINS 

Substances  which  contain  the  protein  molecule  united  to  some  other 
molecule  or  molecules  otherwise  than  as  a  salt. 

(a)  Nucleoproteins. — Compounds  of  one  or  more  protein  molecules 
with  nucleic  acid,  e.g.,  cytoglohulin  from  cytoplasm.  niicIcoJiistone  from 
nucleus. 

(b)  Glycoproteins. — Compounds  of  the  protein  molecule  with  a 
substance  or  substances  containing  a  carbohydrate  group  other  than  a 
nucleic  acid,  e.g.,  mucins  and  mucoids  {osseomucoid  from  bone,  tendomu- 
coid  from  tendon,  ichtlmlin  from  carp  eggs,  helico protein  from  snail). 

(c)  Phosphoproteins. — Compounds  of  the  protein  molecule  with 
some,  as  yet  undefined,  phosphorus-containing  substances  other  than  a 
nucleic  acid  or  lecithin,^  e.g.,  casein  from  milk,  ovovitellin  from  egg 
yolk. 

(d)  Hemoglobins.^ — Compounds  of  the  protein  molecule  with 
hematin,  or  some  similar  substance,  e.g.,  hemoglobin  from  red  blood 
cells,  hemocyanin  from  blood  of  invertebrates. 

(e)  Lecithoproteins. — Compounds  of  the  protein  molecule  with 
lecithins. 

III.  DERIVED  PROTEINS 


I.  Primary  Protein  Derivatives 

Derivatives  of  the  protein  molecule  apparently  formed  through 
hydrolytic  changes  which  involve  only  slight  alteration  of  the  protein 
molecule. 

{a)  Proteans. — Insoluble  products  which  apparently  result  from 
the  incipient  action  of  water,  very  dilute  acids  or  enzymes,  e.g.,  myosan 
from  myosin,  edestan  from  edestin. 

(b)  Metaproteins. — Products  of  the  further  action  of  acids  and  alka- 
lis whereby  the  molecule  is  so  far  altered  as  to  form  products  soluble  in 

^  The  accumulated  chemical  evidence  distinctly  points  to  the  propriety  of  classifying 
the  phosphoproteins  as  conjugated  compounds,  i.e.,  they  are  possibly  esters  of  some  phos- 
phoric acid  or  acids  and  protein. 


PROTEINS  95 

very  weak  acids  and  alkalis  but  insoluble  in  neutral  fluids,  e.g.,  acid 
metaprotein  (acid  albnmiuale),  alkali  mela protein  (alkali  albuminate). 

(c)  Coagulated  Proteins. — Insoluble  products  which  result  from 
(i)  the  action  of  heat  on  their  solutions,  or  (2)  the  action  of  alcohol  on 
the  protein. 

2.  Secondary  Protein  Derivatives^ 

Products  of  the  further  hydrolytic  cleavage  of  the  protein  molecule. 

(0)  Proteoses. — Soluble  in  water,  non-coagulable  by  heat,  and 
precipitated  by  saturating  their  solutions  with  ammonium — or  zinc 
sulphate,^  e.g.,  proto proteose,  deutero proteose. 

(b)  Peptones. — Soluble  in  water,  non-coagulable  by  heat,  but  not 
precipitated  by  saturating  their  solutions  with  ammonium  sulphate,^ 
e.g.,  antipcptone,  amphopeptone. 

(c)  Peptides. — Definitely  characterized  combinations  of  two  or  more 
amino  acids,  the  carboxyl  group  of  one  being  united  with  the  amino 
group  of  the  other  with  the  elimination  of  a  molecule  of  water,^  e.g., 
dipeptides ,  tripeptides,  tetrapeptides,  penta peptides. 

CLASSIFICATION  OF  PROTEINS  ADOPTED  BY  THE  CHEM- 
ICAL AND  PHYSIOLOGICAL  SOCIETIES 
OF  ENGLAND 

I.  Simple  Proteins 
« 

1.  Protamines,  e.g.,  salmine,  clupeine. 

2.  Histones,  e.g.,  globin,  scombrone. 

3.  Albumins,  e.g.,  ovalbumin,  serum  albumin,  vegetable  albumins. 

4.  Globulins,  e.g.,  serum  globulin,  ovoglobulin,  vegetable  globulins. 

5.  Glutelins,  e.g.,  glutenin. 

6.  Alcohol-soluble  proteins,  e.g.,  zein,  gliadin. 

7.  Scleroproteins,  e.g.,  elastin,  keratin. 

8.  Phosphoproteins,  e.g.,  casein,  vitellin. 

II.  Conjugated  Proteins 

1.  Glucoproteins,  e.g.,  mucins,  mucoids. 

2.  Nucleoproteins,  e.g.,  nucleohistone,  cytoglobuUn. 

3.  Chromoproteins,  e.g.,  hemoglobin,  hemocyanin. 

^  The  term  secondary  protein  derivatives  is  used  because  the  formation  of  the  primary 
derivatives  usually  precedes  the  formation  of  the  secondary  derivatives. 

2  As  thus  dctinecl,  this  terna  does  not  strictly  cover  all  the  protein  derivatives  com- 
monly called  i)roleoses,  c.t^.,  hcteroprt)leose  and  dysproteose. 

^  in  this  KTouji  the  kyrines  may  be  included.  I-'or  the  present  it  is  believed  that  it  will 
be  helpful  to  retain  this  term  as  dciined,  reserving  the  expression  peptide  for  the  simpler 
compounds  of  delinite  structure,  such  as  dipeptides,  etc. 

■•  The  peptones  are  undoubtedly  peptides  or  mixtures  of  peptides,  the  latter  term  being 
at  present  used  to  designate  those  of  definite  structure. 


96  PHYSIOLOGICAL   CHEMISTRY 

III.  Products  of  Protein  Hydrolysis 

1.  Infraproteins,  e.g.,  acid  infraprotein  {acid  albuminate),  alkali 
infraprotein  {alkali  albuminate). 

2.  Proteoses,  e.g.,  protoproteose,  hetero proteose,  deutero proteose. 

3.  Peptones,  e.g.,  amphopeptone,  antipeptone. 

4.  Polypeptides,  e.g.,  dipeptides,  tripeptides,  tetrapeptides. 

CONSIDERATIONS  OF  THE  VARIOUS  CLASSES 
OF  PROTEINS 

SIMPLE  PROTEINS 

The  simple  proteins  are  true  protein  substances  which,  upon  hy- 
drolysis, yield  only  a-amino  acids  or  their  derivatives.  "Although 
no  means  are  at  present  available  whereby  the  chemical  individuality  of 
any  protein  can  be  established,  a  number  of  simple  proteins  have  been 
isolated  from  animal  and  vegetable  tissues  which  have  been  so  well 
characterized  by  constancy  of  ultimate  composition  and  uniformity  of 
physical  properties  that  they  may  be  treated  as  chemical  individuals 
until  further  knowledge  makes  it  possible  to  characterize  them  more 
definitely."  Under  simple  proteins  we  may  class  albumins,  globulins, 
gluteHns,  prolamins,  albuminoids,  histones  and  protamines. 

ALBUMINS 

Albumins  constitute  the  first  class  of  simple  proteins  and  may  be 
defined  as  simple  proteins  which  are  coagulable  by  heat  and  soluble 
in  pure  (salt-free)  water.  Those  of  animal  origin  are  not  precipitated 
upon  saturating  their  neutral  solutions  at  30°C.  with  sodium  chloride 
or  magnesium  sulphate,  but  if  a  saturated  solution  of  this  character 
be  acidified  with  acetic  acid  the  albumin  precipitates.  All  albumins 
of  animal  origin  may  be  precipitated  by  saturating  their  solutions  with 
ammonium  sulphate.^  They  may  be  thrown  out  of  solution  by  the 
addition  of  a  sufi&cient  quantity  of  a  mineral  acid,  whereas  a  weak 
acidity  produces  a  slight  precipitate  which  dissolves  upon  agitating  the 
solution.  MetaUic  salts  also  possess  the  property  of  precipitating  al- 
bumins, some  of  the  precipitates  being  soluble  in  excess  of  the  reagent, 
whereas  others  are  insoluble  in  such  an  excess.  Of  those  proteins 
which  occur  native  the  albumins  contain  the  highest  percentage  of  sul- 
phur, ranging  from  1.6  to  2.5  per  cent.     Some  albumins  have  been 

'  In  this  connection,  Osborne's  observation  that  there  are  certain  vegetable  albumins 
which  are  precipitated  by  saturating  their  solutions  with  sodium  chloride  or  magnesium 
sulphate  or  by  half-saturating  with  ammonium  sulphate,  is  of  interest. 


PROTEINS  97 

obtained  in  crystalline  form,  notably  egg  albumin,  serum  albumin,  and 
lactalbumin,  but  the  fact  that  they  may  be  obtained  in  crystalline  form 
does  not  necessarily  prove  them  to  be  chemical  individuals. 

GENERAL  COLOR  REACTIONS  OF  PROTEINS 

These  color  reactions  are  due  to  a  reaction  between  some  one  or 
more  of  the  constituent  radicals  or  groups  of  the  complex  protein  mole- 
cule and  the  chemical  reagent  or  reagents  used  in  any  given  test.  Not 
all  proteins  contain  the  same  groups  and  for  this  reason  the  various  color 
tests  will  yield  reactions  varying  in  intensity  of  color  according  to  the 
nature  of  the  groups  contained  in  the  particular  protein  under  examina- 
tion. Various  substances  not  proteins  respond  to  certain  of  these  color 
reactions,  and  it  is  therefore  essential  to  submit  the  material  under  ex- 
amination to  several  tests  before  concluding  definitely  regarding  its 
nature. 

TECHNIC  OF  THE  COLOR  REACTIONS 

I.  Millon's  Reaction. — To  5  c.c.  of  a  dilute  solution  of  egg  albumin^  in  a  test- 
tube  add  a  few  drops  of  Millon's  reagent.  A  white  precipitate  forms  which  turns 
red  when  heated. 

This  test  is  a  particularly  satisfactory  one  for  use  on  solid  proteins, 
in  which  case  the  reagent  is  added  directly  to  the  solid  substance  and 
heat  applied,  which  causes  the  substance  to  assume  a  red  color.  Such 
proteins  as  are  not  precipitated  by  mineral  acids,  for  example  certain 
of  the  proteoses  and  peptones,  yield  a  red  solution  instead  of  a  red 
precipitate. 

The  reaction  is  due  to  the  presence  of  the  hydroxy-phenyl  group, 
— C6H4OH,  in  the  protein  molecule  and  certain  non-proteins  such  as 
tyrosine,  phenol  (carbolic  acid)  and  thymol  also  respond  to  the  reaction. 
Inasmuch  as  the  tyrosine  grouping  is  the  only  hydroxyphenyl  grouping 
which  has  definitely  been  proven  to  be  present  in  the  protein  molecule  it 
is  evident  that  protein  substances  respond  to  Millon's  reaction  because 
of  the  presence  of  this  tyrosine  complex.  The  test  is  not  a  very  satis- 
factory one  for  use  in  solutions  containing  inorganic  salts  in  large 
amount,  since  the  mercury  of  the  Millon's  reagent-  is  thus  precipitated 
and  the  reagent  rendered  inert.  This  reagent  is  therefore  never  used 
for  the  detection  of  protein  material  in  the  urine.     If  the  solution  under 

1  This  egg  albumin  solution  may  be  prepared  by  beating  egg-white  with  6-10  volumes  of 
water.     The  precipitate  of  ovoglobulin  is  hltered  off  and  the  filtrate  used  in  the  tests. 

2  Millon's  reagent  consists  of  mercury  dissolved  in  nitric  acid  containing  some  nitrous 
acid.  It  is  prepared  by  digesting  one  part  (by  weight)  of  mercury  with  two  parts  (by 
weight)  of  HXO3  (sp.  gr.  1.42)  and  diluting  the  resulting  solution  with  two  volumes  of 
water. 


98  PHYSIOLOGICAL    CHEiUSTRY 

examination  is  strongly  alkaline  it  should  be  neutralized  inasmuch  as 
the  alkali  will  precipitate  yellow  or  black  oxides  of  mercury. 

2.  Xanthoproteic  Reaction. — To  2-3  c.c.  of  egg  albtimin  solution  in  a  test-tube 
add  concentrated  nitric  acid.  A  white  precipitate  forms,  which  upon  heating 
turns  yellow  and  finally  dissolves,  imparting  to  the  solution  a  yeUow  color. 
Cool  the  solution  and  carefully  add  ammonium  hydroxide,  potassium  hydroxide, 
or  sodiimi  hydroxide  in  excess.  Note  that  the  yellow  color  deepens  into  an 
orange. 

This  reaction  is  due  to  the  presence  in  the  protein  molecule  of  the 
phenyl  group — CeHs,  with  which  the  nitric  acid  forms  certain  nitro 
modifications.  The  particular  complexes  of  the  protein  molecule 
which  are  of  especial  importance  in  this  connection  are  those  of  tyrosine, 
phenylalanine,  and  tryptophane.  The  test  is  not  a  satisfactory  one  for 
use  in  urinary  examination  because  of  the  color  of  the  end-reaction. 

3.  GlyoxyUc  Acid  Reaction  (Hopkins-Cole).^ — Place  1-2  c.c.  of  egg  albumin 
solution  and  3  c.c.  of  glyoxyhc  acid,  CHO.COOH  +  H2O  or  CH(0H)2C00H, 
solution  (Hopkins-Cole  reagent")  in  a  test-tube  and  mix  thoroughly.  In  a  second 
tube  place  5  c.c.  of  concentrated  sulphuric  acid.  Incline  the  tube  containing  sul- 
phuric acid  and  by  means  of  a  pipette  allow  the  albvunin-glyoxyUc  acid  solution 
to  flow  carefully  down  the  side.  When  stratified  in  this  manner  a  reddish -violet 
color  forms  at  the  zone  of  contact  of  the  two  fluids. 

This  color  is  due  to  the  presence  of  the  tryptophane  group.  Gelatin 
does  not  respond  to  this  test.  For  formula  of  tryptophane  see  page 
77.  Benedict^  has  suggested  a  new  reagent  for  use  in  carrying  out 
the  Hopkins-Cole  reaction.'^  Nitrates  (XaXOs  and  KXO3)  entirely 
prevent  the  reaction  whereas  formaldehyde  or  nitric  acid  interfere 
somewhat.^ 

4.  Biuret  Test. — To  2-3  c.c.  of  egg  albximin  solution  in  a  test-tube  add  an 
eqiial  volmne  of  concentrated  potassium  hydroxide  solution,  mix  thoroughly, 
and  add  slowly  a  very  dilute  (2-5  drops  in  a  test-tube  of  water)  copper  siilphate 
solution  until  a  purplish-violet  or  pinkish-violet  color  is  produced.     The  depth 

1  Hopkins  and  Cole:  Journal  of  Physiology,  27,  418,  1902. 

-  Hopkins- Cole  reagent  is  prepared  as  follows:  To  one  liter  of  a  saturated  solution  of 
oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  the  mixture  to  stand  until  the 
evolution  of  gas  ceases.     Filter  and  dilute  with  2-3  volumes  of  water. 

^  Benedict:  Journal  of  Biological  Chemistry,  6,  51,  1909. 

*  Benedict's  modified  Hopkins-Cole  reagent  is  prepared  as  follows:  Ten  grams  of  pow- 
dered magnesium  are  placed  in  a  large  Erlenmeyer  flask  and  shaken  up  \\ith  enough  dis- 
tilled water  to  liberally  cover  the  magnesium.  Two  hundred  and  fifty  c.c.  of  a  cold,  satur- 
ated solution  of  oxalic  acid  is  now  added  slowly.  The  reaction  proceeds  very  rapidly  and 
with  the  liberation  of  much  heat,  so  that  the  flask  should  be  cooled  under  running  water 
during  the  addition  of  the  acid.  The  contents  of  the  flask  are  shaken  after  the  addition  of 
the  last  portion  of  the  acid  and  then  poured  upon  a  filter,  to  remove  the  insoluble  magnesium 
oxalate.  A  little  wash  water  is  poured  through  the  filter,  the  filtrate  acidified  with  acetic 
acid  to  prevent  the  partial  precipitation  of  the  magnesium  on  long  standing,  and  made  up  to 
a  liter  with  distilled  water.  This  solution  contains  only  the  magnesium  salt  of  glyoxylic 
acid. 

*  Mathewson:  Dissertation  (Columbia  Univ.),  Eschenbach  Publishing  Co.,  Easton,  Pa., 
1912. 


PROTEINS  99 

of  the  color  depends  upon  the  nature  of  the  protein;  proteoses,  and  peptones 
giving  a  decided  pink,  while  the  color  produced  with  gelatin  is  not  far  removed 
from  a  blue. 

This  reaction  is  given  by  those  substances  which  contain  two  amino 
groups  in  their  molecule,  these  groups  either  being  joined  directly 
together  or  through  a  single  atom  of  nitrogen  or  carbon.  The  amino 
groups  mentioned  must  either  be  two  CONH2  groups  or  one  CONH2 
group  and  one  CSXHo,  C(NH)XH2  or  CH2XH2  group.  It  follows 
from  this  fact  that  substances  which  are  non-protein  in  character  but 
which  contain  the  necessary  groups  will  respond  to  the  biuret  test. 
As  examples  of  such  substances  may  be  cited  oxamide, 

CONH2 

CONH2 

and  biuret, 

CONH2 

\ 
NH. 

/ 
COXH2 

The  test  derives  its  name  from  the  fact  that  this  latter  substance  which 
is  formed  on  heating  urea  to  i8o°C  (see  page  375)  will  respond  to  the 
test.  Protein  material  responds  positively  since  there  are  two  COXH2 
groups  in  the  protein  molecule. 

According  to  Schiff  the  end-reaction  of  the  biuret  test  is  dependent 
upon  the  formation  of  a  copper-potassium-biuret  compound  (cupri- 
potassium  biuret  or  biuret  potassium  cupric  hydroxide).  This  sub- 
stance was  obtained  by  Schiff  in  the  form  of  long  red  needles.  It  has 
the  following  formula: 

OH  OH 

CO  XHa Cu XH2CO 

\  / 

NH  HX 

/  \        . 

CO  •XH2— K       K— NH2CO 

I  I 

OH  OH 

Testing  Colored  Solutions  by  Biuret  Test. — If  the  color  of  the  solu- 
tion is  such  as  to  interfere  with  the  end-reaction  of  the  biuret  test, 
proceed  as  follows:  Make  the  solution  strongly  alkaline  with  potassium 
hydroxide  and  add  a  solution  of  copper  sulphate.  Shake  up  the  mixture 
with  alcohol  and  if  protein  is  present  the  alcohol  will  assume  the  typical 


lOO  PHYSIOLOGICAL    CHEMISTRY 

biuret  coloration.  This  procedure  is  not  applicable  in  case  the  pigment 
of  the  original  solution  is  soluble  in  alcohol.  Excess  of  the  copper  salt 
need  not  be  avoided  in  this  test. 

Gies's  Biuret  Reagent.'^ — Gies  has  devised  a  reagent  for  use  in  the  biuret  test. 
This  reagent  consists  of  lo  per  cent  KOH  solution,  to  which  25  c.c  of  3  per  cent 
CuSOi  solution  per  liter  has  been  added.  This  imparts  a  slight  though  distinct 
blue  color  to  the  clear  liquid.  This  reagent  is  of  material  assistance  in  performing 
the  biuret  test. 

Biuret  Paper  of  Kantor  and  Gies. — According  to  Kantor  and  Gies-  when 
filter  paper  is  immersed  in  the  above  reagent  and  subsequently  dried  it  forms  a 
very' satisfactory-  "biuret  paper"  which  may  be  used  in  a  manner  analogous  to 
indicator  papers.  Moist  papers  may  be  used  in  the  examination  of  powders  which 
are  neutral  or  alkaline  in  reaction.  In  preparing  the  "biuret  paper,"  if  the  filter 
paper  is  left  for  a  sufficient  length  of  time  in  the  reagent  all  traces  of  the  copper 
sulphate  will  be  removed  from  the  solution. 

5.  'Ring  Biuret  Test  (Posner). — This  test  is  particularly  satisfactory  for  use  on 
dilute  protein  solutions,  and  is  carried  out  as  follows.  To  some  dilute  egg  albumin 
in  a  test-tube- add  one-half  its  volume  of  potassium  hydroxide  solution.  Now  hold 
the  tube  in  an  inclined  position  and  allow  some  very  dilute  copper  sulphate  solution, 
made  as  suggested  on  page  98  to  flow  down  the  side,  being  especially  careful  to 
prevent  the  fluids  from  mixing.  At  the  juncture  of  the  two  solutions  the  typical 
end-reaction  of  the  biuret  test  should  appear  as  a  colored  zone  (see  Biuret  Test, 
page  98). 

6.  Liebermann's  Reaction. — Add  about  10  drops  of  concentrated  egg  albumin 
solution  (or  a  little  dry  egg  albumin)  to  about  5  c.c.  of  concentrated  HCl  in  a  test- 
tube.  Boil  the  mixture  until  a  pinkish-violet  color  results.  This  color  was  origi- 
nally supposed  to  indicate  the  presence  of  a  carbohydrate  group  in  the  protein 
molecule,  the  furfural  formed  through  the  action  of  the  acid  upon  the  protein  react- 
ing with  the  hydroxyphenyi  group  of  the  protein  producing  the  pinkish-violet  color. 
It  is  now  considered  uncertain  whether  the  carbohydrate  group  enters  into  the  reac- 
tion. Cole  has  called  attention  to  the  fact  that  a  blue  color  results  if  protein  mate- 
rial which  has  been  boiled  with  alcohol  and  subsequently  washed  with  ether  be  used 
in  making  the  test.  He  believes  the  blue  color  to  be  due  to  an  interaction  between 
the  glyoxylic  acid,  which  was  present  as  an  impurity  in  the  ether  used  in  washing 
the  protein,  and  the  tryptophane  group  of  the  protein  molecule  which  was  split  off 
through  the  action  of  the  acid. 

7.  Acree-Rosenheim  Formaldehyde  Reaction. — Add  a  few  drops  of  a  dilute 
(i  :  5000)  solution  of  formaldehj-de  to  2-3  c.c.  of  egg  albumin  solution  in  a  test-tube. 
Mix  thoroughly  and  after  two  to  three  minutes  carefully  introduce  a  httle  concen- 
trated sulphuric  acid  into  the  tube  in  such  a  manner  that  the  two  solutions  do  not 
mix.  A  violet  zone  will  be  observed  at  the  point  of  juncture  of  the  two  solutions, 
especially  if  the  mixture  is  slightly  agitated.  This  color  probably  results  through 
the  union  of  the  protein  and  the  formaldehyde.  If  the  sulphuric  acid  is  added  to  the 
protein  before  the  formaldehyde  is  added  the  typical  end-reaction  is  not  obtained. 
So  far  as  is  known  this  is  a  specific  test  for  proteins.  The  reaction  cannot  be  applied 
satisfactorily  with  concentrated  formaldehyde. 

1  Gies:  Proceedings  of  Society  of  Biological  Chemists,  Journal  of  Biological  Chemistry, 
7,  60,  1910. 

2  Kantor  and  Gies:  Proc.  Sac.  Biol.  Chem.,  p.  11,  1910. 


PROTEINS  lOI 

Rosenheim  claims  the  reaction  is  due  to  the  presence  of  oxidizing  material  in 
the  sulphuric  acid  and  that  when  pure  sulphuric  acid  is  used  no  reaction  is  obtained. 
He  advises  the  use  of  a  slight  amount  of  an  oxidizing  agent,  e.g.,  ferric  chloride  or 
potassium  nitrate  (0.005  gram  per  100  cc.  of  sulphuric  acid)  in  order  to  facilitate 
the  reaction.  Rosenheim  further  states  that  proteins  respond  to  the  formaldehyde 
reaction  because  of  the  presence  of  the  tryptophane  group,  a  statement  \Yhich  Acree 
does  not  accept  as  proven. 

8.  Bardach's  Reaction.^ — This  is  one  of  the  most  recent  tests  which  have  been 
described  for  the  detection  of  protein  material.  The  test  depends  upon  the  property 
possessed  by  protein  substances  of  preventing  the  formation  of  typical  iodoform 
crystals  through  the  interaction  of  an  alkaline  acetone  solution  with  iodopotassium 
iodide.  Instead  of  the  typical  hexagonal  plates  or  stellar  formations  of  iodoform 
there  are  produced,  under  the  conditions  of  the  test,  fine  yelloiv  needles  which  are 
apparently  some  iodine  compound  other  than  iodoform.  The  technic  of  the  test 
is  as  follows:  Place  about  5  cc.  of  the  protein  solution-  under  examination  in  a  test- 
tube,  add  2-3  drops  of  a  0.5  per  cent  solution  of  acetone  and  sufficient  Lugol's  solu- 
tion' to  supply  a  moderate  excess  of  iodine  and  produce  a  red-brown  coloration. 
(The  amount  of  Lugol's  solution  necessary  will  depend  upon  the  content  of  protein, 
sugar,  and  other  iodine-reacting  substances  in  the  solution  under  examination  and 
may  vary  from  one  drop  to  several  cubic  centimeters.)  Add  an  excess  (ordinarily 
about  3  CO.)  of  concentrated  ammonium  hydroxide  and  thoroughly  mix  the  solu- 
tion. Place  the  tube  in  the  test-tube  rack,  examine  the  contents  at  intervals  of 
five  minutes,  and  when  it  is  evident  that  crystals  have  formed,  place  a  drop  of  the 
mixture  upon  a  microscopic  slide,  put  a  cover-glass  in  position,  and  examine  the 
mixture  under  the  microscope.  The  formation  of  canary  yellow  crystals  indicates 
the  presence  of  protein  material  in  the  solution  examined.  The  crystals  are  ordi- 
narily needle-like  in  appearance  and  show  a  tendency  to  assume  rosette  or  bundle- 
like formations,  but  under  certain  conditions  they  may  show  knobbed  (nail-like) 
and  branching  variations. 

If  a  moderate  excess  of  iodine  is  used  in  making  the  test,  a  black  precipitate 
of  iodonitro  compounds  is  at  once  formed  upon  the  addition  of  the  ammonium 
hydroxide,  and  yellow  needles  are  subsequently  deposited  upon  it.  In  case  just  the 
proper  amount  of  iodine  is  used,  the  solution  soon  assumes  a  yellow  color  and  the 
black  precipitate  formed  upon  the  addition  of  the  ammonium  hydroxide  is  gradually 
transformed  more  or  less  completely  into  the  yellow  cr>'stals.  In  either  case  the 
needles  ordinarily  form  within  an  hour,  and  frequently  in  a  much  shorter  time. 
If  too  great  an  excess  of  iodine  is  employed  the  heavy  black  precipitate  may  obscure 
or  even  prevent  the  reaction.  The  presence  of  insufficient  iodine  or  excess  protein 
may  likewise  prevent  the  reaction.  In  tests  in  which  a  concentrated  protein  solu- 
tion and  an  excess  of  iodine  are  used,  the  addition  of  ammonium  hydroxide  im- 
mediately produces  a  grayish-green  precipitate.  In  such  instances,  if  the  propor- 
tions are  favorable  and  the  mixture  be  stirred  with  a  glass  rod  for  a  few  minutes, 
the  precipitate  is  gradually  transformed  into  the  crystals  before  mentioned. 

It  is  probable  that  all  soluble  proteins  will  respond  to  Bardach's  reaction,  but 
the  relative  delicacy  of  the  reaction  as  well  as  the  value  of  the  test  as  compared  with 

^Bardach:  Zeilschrifl  fiir  Pliysiologisclic  Chcmic,  54,  355,  1908;  also  Seaman  and  Gies: 
Proceedings  of  the  Society  for  Experinictital  Biology  and  Medicine,  5,  125,  190S. 
-  The  solution  should  not  contain  more  than  5  per  cent  of  protein  material. 
^  Dissolve  4  grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  cc.  of  distilled  water. 


I02  PHYSIOLOGICAL   CHEMISTRY 

other  protein  tests  remain  to  be  determined.     The  only  disturbing  factor  noted 
thus  far  is  the  presence  of  earthy  phosphates  in  the  solution  under  examination. 

PRECIPITATION  REACTIONS  AND  OTHER  PROTEIN  TESTS 

There  are  three  forms  in  which  proteins  may  be  precipitated,  i.e., 
unaltered,  as  an  albuminate,  and  as  an  insoluble  salt.  An  instance  of  the 
precipitation  in  a  native  or  unaltered  condition  is  seen  in  the  so-called 
salting-out  experiments.  Various  salts,  notably  (NH4)2S04,  ZnS04, 
MgS04,  Na2S04  and  NaCl,  possess  the  power,  when  added  in  solid  form  to 
certain  definite  protein  solutions,  of  rendering  the  menstruum  incapable 
of  holding  the  protein  in  solution,  thereby  causing  the  protein  to  be 
precipitated  or  salted-out,  to  use  the  common  term.  Mineral  acids  and 
alcohol  also  precipitate  proteins  unaltered.  In  the  case  of  concentrated 
acids  the  protein  is  dissolved  in  the  presence  of  an  excess  of  acid  with 
the  formation  of  a  protein  salt.  Proteins  are  precipitated  as  albu- 
minates when  treated  with  certain  metallic  salts,  and  precipitated  as 
insoluble  salts  when  weak  organic  acids  such  as  certain  of  the  alkaloidal 
reagents  are  added  to  their  solutions. 

If  certain  acids  (picric,  phosphotungstic,  phosphomolybdic,  tannic,  or 
chromic)  be  added  to  a  neutral  albumin  solution  a  precipitate  of  an 
insoluble  protein  salt  occurs.  If,  however,  the  salts  of  these  acids  be 
added  no  precipitate  occurs.  The  addition  of  a  small  amount  of  acid, 
as  acetic  acid,  to  such  a  solution  will  cause  a  precipitate  to  form.^ 

The  effect  of  the  addition  of  the  salts  of  the  heavy'metals  is  in  the 
first  instance  to  cause  a  precipitation  of  the  protein.  In  many  cases, 
however,  the  addition  of  an  excess  of  such  salts  causes  the  solution  of 
the  precipitate  while  a  further  excess  may  cause  a  reprecipitation.  The 
precipitate  which  is  first  formed  in  a  protein  solution  by  the  addition 
of  the  salts  of  the  heavy  metals  may  be  redissolved  not  only  by  an 
excess  of  such  salts  but  by  an. excess  of  protein  as  well.^ 

It  is  generally  stated  that  globulins  are  precipitated  from  their  solu- 
tions upon  half  saturation  with  ammonium  sulphate  and  that  albumins 
are  precipitated  upon  complete  saturation  by  this  salt.  Comparatively 
few  exceptions  were  found  to  this  rule  until  proteins  of  vegetable  origin 
came  to  be  more  extensively  studied.  These  studies,  furthered  es- 
pecially by  Osborne  and  associates,  have  demonstrated  very  clearly 
that  the  characterization  of  a  globulin  as  a  protein  which  is  precipitated 
by  half  saturation  with  ammonium  sulphate,  can  no  longer  hold. 
Certain  vegetable  globuHns  have  been  isolated  which  are  not  precipi- 

1  Mathews:  Amer.  Jour,  of  Physiology,  i,  445,  1898. 

^Pauli:  Hojmeisler's  Beitrage,  6,233,   ^9°A~°S'>  Robertson:  Ergehnisse  der  Physiologic, 
ro,  290,  1910. 


PROTEINS  103 

tated  by  this  salt  until  a  concentration  is  reached  greater  than  that 
secured  by  half-saturation.  As  an  example  of  an  albumin  which  does 
not  conform  to  the  definition  of  an  albumin  as  regards  its  precipitation 
by  ammonium  sulphate  may  be  mentioned  the  leucosin  of  the  wheat 
germ,  which  is  precipitated  from  its  solution  upon  /?o//-saturation  with 
ammonium  sulphate.  The  limits  of  precipitation  by  ammonium 
sulphate,  therefore,  do  not  furnish  a  sufficiently  accurate  basis  for  the 
differentiation  of  globuUns  from  albumins.  It  has  further  been  deter- 
mined that  a  given  protein  which  is  precipitable  by  ammonium  sulphate 
cannot  be  ''salted-out"  by  the  same  concentration  of  the  salt  under  all 

conditions. 

Experiments 

1.  Influence  of  Concentrated  Mineral  Acids,  Alkalis  and  Organic  Acids. — 
Prepare  five  test-tubes  each  containing  5  c.c.  of  concentrated  egg  albumin  solu- 
tion. To  the  first  add  concentrated  HjS04,  drop  by  drop,  until  an  excess  of  the 
acid  has  been  added.  Note  any  changes  which  may  occur  in  the  solution.  AUow 
the  tube  to  stand  for  24  hours  and  at  the  end  of  that  period  observe  any  altera- 
tion which  may  have  taken  place.  Heat  the  tube  and  note  any  further  change 
which  may  occur.  Repeat  the  experiment  in  the  four  remaining  tubes  with 
concentrated  hydrochloric  acid,  concentrated  nitric  acid,  concentrated  potassium 
hydroxide  and  acetic  acid.  How  do  strong  mineral  acids,  strong  alkalis,  and 
strong  organic  acids  differ  in  their  action  toward  protein  solutions? 

2.  Precipitation  by  Metallic  Salts. — Prepare  four  tubes  each  containing  2-3 
c.c.  of  dilute  egg  albumin  solution.  To  the  first  add  mercuric  chloride,  drop  by 
drop  slowly,  until  an  excess  of  the  reagent  has  been  added,  noting  any  changes 
which  may  occur.  If  not  added  very  gradually  the  formation  of  the  precipitate 
may  not  be  noted,  due  to  its  solubiUty  in  excess  of  the  reagent.  Repeat  the  ex- 
periment with  lead  acetate,  silver  nitrate,  copper  sulphate,  ferric  chloride,  and 
bariimi  chloride,  using  very  dilute  solutions. 

Egg  albumin  is  used  as  an  antidote  for  lead  or  mercury  poisoning. 
Why  ?     Is  it  an  equally  good  antidote  for  the  other  metalUc  salts  tested  ? 

3.  Precipitation  by  Alkaloidal  Reagents. — Prepare  six  tubes  each  containing 
2-3  c.c.  of  dilute  egg  albumin  solution.  To  the  first  add  picric  acid  drop  by  drop 
imtil  an  excess  of  the  reagent  has  been  added,  noting  any  changes  which  may 
occur.  Repeat  the  experiment  with  trichloracetic  acid,  tannic  acid,  phospho- 
timgstic  acid,  phosphomolybdic  acid,  and  potassio-mercuric  iodide.  Are  these 
precipitates  soluble  in  excess  of  the  reagent?  Acidify  with  hydrochloric  acid 
before  testing  with  the  last  three  reagents. 

4.  Nitric  Acid  Test  (Heller).— Place  5  c.c.  of  concentrated  nitric  acid  in  a 
test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  dilute  albimiin 
solution  to  flow  slowly  down  the  side.  The  Uquids  should  stratify  with  the 
formation  of  a  white  zone  of  precipitated  albxmiin  at  the  point  of  juncture.  This 
is  a  very  delicate  test  and  is  further  discussed  on  page  423. 

An  apparatus  called  the  albttmoscope  or  Itorismoscope  has  been  devised  for  use 
in  the  tests  of  this  character  and  has  met  with  considerable  favor.  The  method  of 
using  the  albumoscope  is  described  on  p.  104.  The  instrument  is  shown  in  Fig. 
130,  p.  424. 


I04  PHYSIOLOGICAL   CHEMISTRY 

Use  of  the  Alhumoscope. — This  instrument  is  intended  to  facilitate  the  making  of 
"ring"  tests  such  as  Heller's  and  Roberts'.  In  making  a  test  about  $  c.c.  of  the 
solution  under  examination  is  first  introduced  into  the  apparatus  through  the  larger 
arm  and  the  reagent  used  in  the  particular  test  is  then  introduced  through  the  capil- 
lary arm  and  allowed  to  flow  down  underneath  the  solution  under  examination. 
If  a  reasonable  amount  of  care  is  taken  there  is  no  possibility  of  mixing  the  two  solu- 
tions and  a  definitely  defined  white  "ring"  is  easily  obtained  at  the  zone  of  contact. 

5.  Nitric  Acid — MgS04  Test  (Roberts). — Place  5  c.c.  of  Roberts'  reagent^  in 
a  test-tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  albumin  solu- 
tion to  flow  slowly  down  the  side.  The  liquids  should  stratify  with  the  formation 
of  a  white  zone  of  precipitated  albumin  at  the  point  of  juncture.  This  test  is  a 
modification  of  Heller's  ring  test  and  is  rather  more  satisfactory.  The  alhumo- 
scope may  also  be  used  in  making  this  test  (see  Fig.  130,  page  424). 

6.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent^  in  a  test-tube,  in- 
cline the  tube,  and  by  means  of  a  pipette  allow  5  c.c.  of  albumin  solution,  acidified 
with  acetic  acid,  to  flow  slowly  down  the  side.  A  white  zone  will  form  at  the  point 
of  contact.  This  is  an  exceedingly  delicate  test,  in  fact  too  delicate  for  ordinary 
clinical  purposes,  since  it  serves  to  detect  albumin  when  present  in  the  merest  trace 
(i  :  250,000).     This  test  is  further  discussed  on  page  424. 

7.  Tanret's  Test. — To  5  c.c.  of  albumin  solution  in  a  test-tube  add  Tanret's 
reagent,'  drop  by  drop,  until  a  turbidity  or  precipitate  forms.  This  is  an  exceed- 
ingly delicate  test.  Sometimes  the  albumin  solution  is  stratified  upon  the  reagent 
as  in  Heller's  or  Roberts'  ring  tests.  In  urine  examination  it  is  claimed  by  Repiton 
that  the  presence  of  urates  lowers  the  delicacy  of  the  test.  Tanret  claims  that  the 
removal  of  urates  is  not  necessary  inasmuch  as  the  urate  precipitate  will  disappear 
on  warming  and  the  albumin  precipitate  will  not.  He  says,  however,  that  mucin 
interferes  with  the  delicacy  of  his  test  and  should  be  removed  by  acidification  with 
acetic  acid  and  filtration  before  testing  for  albumin. 

8.  Sodium  Chloride  and  Acetic  Acid  Test.^ — Mix  2  volumes  of  albumin  solu- 
tion and  I  volume  of  a  saturated  solution  of  sodium  chloride  in  a  test-tube,  acidify 
with  acetic  acid,  and  heat  to  boihng.  The  production  of  a  cloudiness  or  the 
formation  of  a  precipitate  indicates  the  presence  of  albumin. 

9.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c.  of  dilute  egg 
albumin  solution  in  a  test-tube  add  5-10  drops  of  acetic  acid.  Mix  well  and 
add  potassium  ferrocyanide,  drop  by  drop,  until  a  precipitate  forms.  This  test 
is  very  delicate. 

Schmiedl  claims  that  a  precipitate  of  Fe(Cn)6K2Zn  or  Fe(Cn)6- 
Zn2,  is  formed  when  solutions  containing  zinc  are  subjected  to  this  test, 
and  that  this  precipitate  resembles  the  precipitate  secured  with  protein 

1  Roberts'  reagent  is  composed  of  i  volume  of  concentrated  HNO3  and  5  volumes  of  a 
saturated  solution  of  MgS04. 

2  Spiegler's  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Sodium  chloride.. 50  grams. 

Glycerol 100  grams. 

Distilled  water 1000  grams. 

'  Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  grams  of  mercuric  chloride  in  25 
c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide  dissolved  in  25  c.c.  of 
water,  then  make  the  total  solution  up  to  60  c.c.  with  water  and  add  20  c.c.  of  glacial  acetic 
acid  to  the  combined  solutions. 


PROTEINS  105 

solutions.  In  the  case  of  human  urine  a  reaction  was  obtained  when 
0.000022  gram  of  zinc  per  cubic  centimeter  was  present.  Schmiedl 
further  found  that  the  urine  collected  from  rabbits  housed  in  zinc-lined 
cages  possessed  a  zinc  content  which  was  sufficient  to  yield  a  ready  re- 
sponse to  the  test.  Zinc  is  the  only  interfering  substance  so  far 
reported. 

10.  Salting-out  Experiments. — (a)  To  25  c.c.  of  egg  albumin  solution  in  a 
small  beaker  add  solid  ammonium  sulphate  to  the  point  of  saturation,  keeping 
the  temperature  of  the  solution  below  40°C.  Filter,  test  the  precipitate  by 
Millon's  reaction  and  the  filtrate  by  the  biuret  test.  What  are  your  conclu- 
sions? (b)  Repeat  the  above  experiment,  making  the  saturation  with  solid 
sodium  chloride.  How  does  this  result  differ  from  the  result  of  the  saturation 
with  ammonium  sulphate?     Add  2-3  drops  of  acetic  acid.     What  occurs? 

All  proteins  except  peptones  are  precipitated  by  saturating  their 
solutions  with  ammonium  sulphate.  Globulins  are  the  only  proteins 
precipitated  by  saturating  with  sodium  chloride  (see  Globulins,  page 
108),  unless  the  saturated  solution  is  subsequently  acidified,  in  which 
event  all  proteins  except  peptones  are  precipitated. 

Soaps  may  be  salted-out  in  a  similar  manner  (see  page  181). 

11.  Coagulation  or  Boiling  Test. — Heat  25  c.c.  of  dilute  egg  albumin  solution 
to  the  boiling-point  in  a  small  evaporating  dish.  The  albumin  coagulates.  Com- 
plete coagulation  may  be  obtained  by  acidifying  the  solution  with  3-5  drops  of 
acetic  acid^  at  the  boiling-point.     Test  the  coagulimi  by  Millon's  reaction. 

The  acid  is  added  to  neutralize  any  possible  alkalinity  of  the  solu- 
tion, to  dissolve  any  substances  which  are  not  albumin  and  to  facilitate 
coagulation  (see  further  discussion  on  pages  117  and  424). 

12.  Coagulation  Temperature. — Prepare  four  test-tubes  each  containing  5  c.c 
of  neutral  egg  albumin  solution.  To  the  first  add  i  drop  of  0.2  per  cent  hydro- 
chloric acid,  to  the  second  add  i  drop  of  0.5  per  cent  sodivmi  carbonate  solution, 
to  the  third  add  i  drop  of  10  per  cent  sodium  chloride  solution  and  leave  the 
fourth  neutral  in  reaction.  Partly  fill  a  beaker  of  medium  size  with  water  and 
place  it  within  a  second  larger  beaker  which  also  contains  water,  the  two  vessels 
being  separated  by  pieces  of  cork.  Fasten  the  four  test-tubes  compactly  together 
by  means  of  a  rubber  band,  lower  them  into  the  water  of  the  inner  beaker  and 
suspend  them,  by  means  of  a  clamp  attached  to  one  of  the  tubes,  in  such  a  manner 
that  the  albumin  solutions  shall  be  midway  between  the  upper  and  lower  sur- 
faces of  the  water.  In  one  of  the  tubes  place  a  thermometer  with  its  bulb  entirely 
beneath  the  surface  of  the  albumin  solution  (Fig.  36).  Gently  heat  the  water  in 
the  beakers,  noting  carefully  any  changes  which  may  occur  in  the  albumin  solu- 
tions and  record  the  exact  temperature  at  which  these  changes  occur.  The 
first  appearance  of  an  opacity  in  an  albimiin  solution  indicates  the  commencement 
of  coagulation  and  the  temperature  at  which  this  occurs  should  be  recorded  as 
the  coagulation  temperature  for  that  particular  albumin  solution. 

1  Nitric  acid  is  often  used  in  place  of  acetic  acid  in  this  test.  In  case  nitric  acid  is  used, 
ordinarily  1-2  drops  are  sutlicient. 


io6 


PHYSIOLOGICAL   CHEMISTRY 


What  is  the  order  in  which  the  four  solutions  coagulate? 
Repeat  the  experiment,  adding  to  the  first  tube  i  drop  of  acetic  acid,  to  the 
second  i  drop  of  concentrated  potassium  hydroxide  solution,  to  the  third  2  drops 
of  a  10  per  cent  sodium  chloride  solution  and  leave  the  fourth  neutral  as  before. 
What  is  the  order  of  coagulation  here?    Why?     See  page  116. 
13.  Precipitation   by  Alcohol. — Prepare  three   test-tubes   each   containing 
about  10  c.c.  of  95  per  cent  alcohol.     To  the  first  add  i  drop  of  0.2  per  cent 

hydrochloric  acid,  to  the  second  i  drop  of  potas- 
siiun  hydroxide  solution  and  leave  the  third 
neutral  in  reaction.  Add  to  each  tube  a  few 
drops  of  egg  albmnin  solution  and  note  the  re- 
sults. What  do  you  conclude  from  this  experi- 
ment? 


OB 


If  in  acid  or  neutral  solution  alcohol 
precipitates  proteins  unaltered,  but  if  al- 
lowed to  remain  under  alcohol  the  protein  is 
transformed.  The  "fixing"  of  tissues  for 
histological  examination  by  means  of  al- 
cohol is  an  illustration  of  the  application 
of  this  transformation  produced  by  alcohol. 
It  apparently  is  a  process  of  dehydration. 

14.  CrystaUization  of  Egg  Albmnin.  ^ — Care- 
fully remove  the  egg-white  from  a  number  of 
absolutely  fresh  eggs.^  Measure  the  volume  of 
the  egg-white  and  add  an  equal  volume  of  satur- 
ated ammonium  sulphate  a  small  portion  at  a 
time,  beating  the  mixture  vigorously  after  each 
addition.^  Filter  the  mixture  through  a  large 
pleated  filter  paper.*  Measure  the  volume  of 
the  filtrate.  To  100  c.c.  of  the  filtrate  add  very 
carefully  a  10  per  cent  solution  of  acetic  acid  from 
a  burette  being  certain  to  note  the  exact  volume 
of  the  acid  used.  The  acid  should  be  added  drop 
by  drop,  the  albumin  mixture  being  gently  shaken 
during  the  process.  Add  acid  until  the  precipi- 
tate_  which  forms  at  each  addition  is  no  longer  dissolved  when  the  albumin  is 
shaken,  and  an  opalescent  mixture  is  secured.  (It  is  generally  rather  difiicult 
to  determine  this  point,  inasmuch  as  suspended  air  bubbles  may  simulate  a 
precipitate.)  As  soon  as  the  solution  is  milky,  indicating  that  a  permanent  pre- 
cipitate has  formed,  run  in  from  the  burette  i  cc.  of  the  acetic  acid.  This  should 
produce  a  heavy  white  precipitate.  Now  ^ake  the  burette  reading  to  determine 
the  exact  volume  of  acid  used  in  the  treatment  of  100  c.c  of  the  albumin  mixture. 

^  Hopkins  and  Pinkus:  Jour.  Physiol.,  23. 
2  If  not  perfectly  fresh  the  albumin  will  not  crystallize. 
^  Note  the  odor  of  ammonia.     What  causes  it? 

*  Sometimes  better  results  are  obtained  by  permitting  the  mixture  to  stand  several 
hours  before  filtering. 


Fig.  36. — Coagulation  Tempera- 
ture Apparatus. 


PROTEINS  107 

Calculate  the  exact  volume  of  acid  necessary  to  precipitate  the  remaining  portion 
of  the  original  albumin  mixture  and  add  this  calculated  quantity.  Mix  the  two 
portions  of  albumin  and  allow  to  stand  over  night.  Remove  a  drop  of  the  suspended 
material  to  a  slide  and  examine  microscopically.  Crystals  in  the  form  of  fine 
needles  will  be  observed.  This  is  the  crystallized  egg  albumin.  To  recrystallize, 
filter  off  the  crystals  and  dissolve  them  in  the  smallest  possible  volume  of  water. 
Filter,  and  to  the  filtrate  carefuUy  add  saturated  ammonium  sulphate  until  a  faint, 
permanent  precipitate  is  formed.  Allow  the  mixture  to  stand  several  hours  and 
examine  as  before.  The  crystals  of  albumin  should  be  somewhat  larger  than  when 
first  examined. 

The  above  method  may  also  be  used  for  crystallizing  serum  albumin  from  the 
fresh  blood  serum  of  the  horse,  mule  or  ass. 

15.  Preparation  of  Powdered  Egg  Albumin. — This  may  be  prepared  as  follows: 
Ordinary  egg-white  finely  divided  by  means  of  scissors  or  a  beater  is  treated  with 
4  volumes  of  water  and  filtered.  The  filtrate  is  evaporated  on  a  water-bath  at 
about  5o°C.  and  the  residue  powdered  in  a  mortar. 

16.  Tests  on  Powdered  Egg  Albumin. — ^With  powdered  albumin  prepared  as 
described  above  (by  yourself  or  furnished  by  the  instructor),  try  the  following 
tests : 

(a)  Solubility .^ — Test  the  solubility  of  the  albumin  in  water,  sodium  chloride, 
dilute  acid  and  alkali. 

(b)  Millon's  Reaction. 

(c)  Glyoxylic  Acid  Reaction  (Hopkins-Cole). — ^When  used  to  detect  the 
presence  of  protein  in  solid  form  this  reaction  should  be  conducted  as  follows : 
Place  5  c.c.  of  concentrated  sulphuric  acid  in  a  test-tube  and  add  carefiilly,  by 
means  of  a  pipette,  3-5  c.c.  of  HopMns-Cole  reagent.  Introduce  a  small  amount 
of  the  solid  substance  to  be  tested,  agitate  the  tube  sUghtly,  and  note  that  the 
suspended  pieces  assimie  a  reddish-violet  color,  which  is  the  characteristic  end- 
reaction  of  the  Hopkins-Cole  test ;  later  the  solution  will  also  assume  the  reddish- 
violet  color. 

(d)  Composition  Test. — Heat  some  of  the  dry  powder  in  a  dry  test-tube  in 
which  is  suspended  a  strip  of  moistened  red  Utmus  paper  and  across  the  mouth  of 
which  is  placed  a  piece  of  filter  paper  moistened  with  lead  acetate  solution. 
As  the  powder  is  heated  it  chars,  indicating  the  presence  of  carbon;  the  fimies  of 
ammonia  are  evolved,  tiurning  the  red  litmus  paper  blue  and  indicating  the  pres- 
ence of  nitrogen  and  hydrogen;  the  lead  acetate  paper  is  blackened,  indicating 
the  presence  of  sulphur,  and  the  deposition  of  moisture  on  the  side  of  the  tube 
indicates  the  presence  of  hydrogen.  Moisture  indicates  hydrogen  only  in  case 
both  powder  and  test-tube  used  in  the  test  are  absolutely  dry. 

(e)  Coagulation  Test. — Immerse  a  dry  test-tube  containing  a  Uttle  powdered 
egg  albumin  in  boiling  water  for  a  few  moments.  Remove  and  test  the  solubUity 
of  the  albvmiin  according  to  the  directions  given  under  (ai  above.  It  is  still 
soluble.  Why  has  it  not  been  coagulated?  Repeat  the  above  experiments 
with  powdered  serimi  albumin  and  see  how  the  results  compare  with  those 
just  obtained. 

SULPHUR  IX  PROTEIN 

Sulphur  is  believed  to  be  present  in  two  different  forms  in  the  pro- 
tein molecule.     The  first  form,  which  is  present  in  greatest  amount, 


Io8  PHYSIOLOGICAL    CHEMISTRY 

is  that  loosely  combined  wdth  carbon  and  hydrogen.  An  example  of 
this  combination  is  shown  in  cystine, 

CH2"S — SCH2 

I  I 

CHXH2    CHNH2 

I  I 

COOH       COOH 

Sulphur  in  this  form  is  variously  termed  unoxidized,  loosely  combined, 
mercaptan,  and  lead-hlackening  sulphur.  The  second  form  is  combined 
in  a  more  stable  manner  with  carbon  and  oxygen  and  is  known  as 
oxidized  or  acid  sulphur.  The  protamines  are  the  only  class  of  sulphur- 
free  proteins. 

Tests  for  Sulphur 

1.  Tests  for  Unoxidized  Sulphur. — (a)  To  equal  volumes  of  KOH  and  egg 
albumin  solutions  in  a  test-tube  add  1-2  drops  of  lead  acetate  solution  and  boil  the 
mixture.  Unoxidized  sulphur  is  indicated  by  a  darkening  of  the  solution,  the 
color  deepening  into  a  black  if  sufficient  sulphur  is  present.  Add  hydrochloric 
acid  and  note  the  characteristic  odor  evolved  from  the  solution.  Write  the  reac- 
tions for  this  test,  (b)  Place  equal  volxmies  of  KOH  and  egg  albximin  solutions 
in  a  test-tube  and  boil  the  mixture  vigorously.  Cool,  make  acid  with  glacial 
acetic  acid  and  add  1-2  drops  of  lead  acetate.  A  darkening  indicates  the  pres- 
ence of  vmoxidized  sulphur. 

2.  Test  for  Total  Sulphur  (Unoxidized  and  Oxidized). — Place  the  substance 
to  be  examined  (powdered  egg  albmnin)  in  a  small  porcelain  crucible,  add  a  suit- 
able amount  of  soUd  fusion  mixture  (sodiimi  carbonate  and  potassium  nitrate 
mixed  in  the  proportion  2:1)  and  heat  carefully  until  a  colorless  mixture  results. 
(Sodium  peroxide  may  be  used  in  place  of  this  fusion  mixture  if  desired.)  Cool, 
dissolve  the  cake  in  a  Uttle  warm  water  and  filter.  Acidify  the  filtrate  with  hydro- 
chloric acid,  heat  it  to  the  boiUng-point  and  add  a  small  amount  of  barium  chlor- 
ide solution.  A  white  precipitate  forms  if  sulphur  is  present.  What  is  this 
precipitate? 

GLOBULINS 

Globulins  are  simple  proteins  especially  predominant  in  the  vege- 
table kingdom.  They  are  closely  related  to  the  albumins  and  in  com- 
mon with  them  give  all  the  ordinary  protein  tests.  Globulins  dififer 
from  the  albumins  in  being  insoluble  in  pure  (salt-free)  water.  They 
are,  however,  soluble  in  neutral  solutions  of  salts  of  strong  bases  with 
strong  acids.  Most  globulins  are  precipitated  from  their  solutions  by 
saturation  with  solid  sodium  chloride  or  magnesium  sulphate.  As  a 
class  they  are  much  less  stable  than  the  albumins,  a  fact  shown  by  the 
increasing  difl&culty  with  which  a  globulin  dissolves  during  the  course  of 
successive  reprecipitations. 

We  have  used  an  albumin  of  animal  origin  (egg  albumin),  for  all 


PROTEINS  109 

the  protein  tests  thus  far,  whereas  the  globuHn  to  be  studied  will  be 
prepared  from  a  vegetable  source.  There  being  no  essential  difiference 
between  animal  and  vegetable  proteins,  the  vegetable  globulin  we  shall 
study  may  be  taken  as  a  true  type  of  all  globulins,  both  animal  and 
vegetable. 

Experiments  on  Globulin 

Preparation  of  the  Globulin.^ — Extract  20-30  grams  fa  handful)  of  crushed 
hemp  seed  with  a  5  per  cent  solution  of  sodium  chloride  for  one-half  hour  at 
6o°C.  Filter  while  hot  through  a  paper  moistened  with  5  per  cent  sodium  chloride 
solution.  Place  the  filtrate  in  the  water-bath  at  6o'C.  and  allow  it  to  stand  for 
24  hours  in  order  that  the  globuUn  may  crystaUize  slowly.  In  case  the  filtrate  is 
cloudy  it  should  be  warmed  to  6o'C.  in  order  to  produce  a  clear  solution.  The 
globuUn  is  soluble  in  hot  5  per  cent  sodium  chloride  solution  and  is  thus  extracted 


Fig.  37. — Edestix. 

from  the  hemp  seed,  but  upon  cooling  this  solution  much  of  the  globulin  separates 
in  crystalline  form.  This  particular  globulin  is  called  edestin.  It  crystaUizes 
in  several  different  forms,  chiefly  octahedra  isee  Fig.  37,  above).  (The  crystal- 
line form  of  excelsin,  a  protein  obtained  from  the  Brazil  nut,  is  shown  in  Fig.  38, 
p.  no.  This  vegetable  protein  crystaUizes  in  the  form  of  hexagonal  plates.) 
Filter  off  the  edestin  and  make  the  following  tests  on  the  crystaUine  body  and  on 
the  filtrate  which  still  contains  some  of  the  extracted  globulin. 

Tests  on  Crystallized  Edestin. — Microscopical  examination  (see  Fig.  37. 

(2)  SolubiUty. — Try  the  solubihty  in  the  ordinary  solvents  i.see  page  21). 
Keep  these  solubiUties  in  mind  for  comparison  with  those  of  edestan,  to  be  made 
later  (see  page  115). 

(3)  Millon's  Reaction. 

(4)  Coagiilation  Test. — Place  a  small  amount  of  the  globuhn  in  a  test-tube,  add 
a  little  water  and  boil.  Now  add  dilute  hydrochloric  acid  and  note  that  the  pro- 
tein no  longer  dissolves.     It  has  been  coagiilated. 


no 


PHYSIOLOGICAL   CHEMISTRY 


(5)  Dissolve  the  remainder  of  the  edestin  in  0.2  per  cent  hydrochloric  acid 
and  preserve  this  acid  solution  for  use  in  the  experiments  on  proteans  (see  page 

115)- 

Tests  on  Edestin  Filtrate. — (i)  Influence  of  Protein  Precipitants. — ^Try  a 
few  protein  precipitants  such  as  nitric  acid,  tannic  acid,  picric  acid,  and  mercuric 
chloride. 

(2)  Biuret  Test. 

(3)  Coagulation  Test. — ^Boil  some  of  the  filtrate  in  a  test-tube.  What 
happens? 

(4)  Saturation  with  Sodium  Chloride.^Saturate  some  of  the  filtrate  with 
solid  sodium  chloride.  How  does  this  result  differ  from  that  obtained  upon 
saturating  egg  albumin  solution  with  solid  sodium  chloride? 


Fig.  38. — ExcELSiN,  The  Protein  of  the  Brazil  Nut. 
(Drawn  from  crystals  furnished  by  Dr.  Thomas  B;  Osborne,  New  Haven,  Conn.) 

(5)  Precipitation  by  Dilution. — Dilute  some  of  the  filtrate  with  10-15  volumes 
of  water.    Why  does  the  globuhn  precipitate? 


Glutelins 

It  has  been  repeatedly  shown,  particularly  by  Osborne,  that  after 
extracting  the  seeds  of  cereals  with  water,  neutral  salt  solution,  and 
strong  alcohol,  there  still  remains  a  residue  which  contains  protein 
material  which  may  be  extracted  by  very  dilute  acid  or  alkali.  These 
proteins  which  are  insoluble  in  all  neutral  solvents,  but  readily  soluble 
in  very  dilute  acids  and  alkalis  are  called  glutelins.  The  only  member 
of  the  group  which  has  yet  received  a  name  is  the  glutenin  of  wheat, 
a  protein  which  constitutes  nearly  50  per  cent  of  the  gluten,  the  re- 
mainder being  principally  gliadin.  It  is  not  definitely  known  whether 
glutelins  occur  as  constituents  of  all  seeds. 


PROTEINS  III 

Gluten:  Preparation  and  Tests. ^ — To  about  50  grams  of  wheat  flour  in  a 
casserole  or  evaporating  dish,  add  a  little  water  and  mix  thoroughly  until  a  stiff 
dough  results.  Knead  this  dough  thoroughly  and  permit  it  to  stand  for  about  a 
half  hour.  This  is  done  in  order  that  the  maximum  quantity  of  gluten  may  be 
obtained.  Treat  the  dough  with  about  200  c.c.  of  water  and  knead  it  thoroughly. 
Note  the  yellowish  color  of  the  dough  and  the  milky  appearance  of  the  water  due 
to  suspended  starch  granules.  (Place  a  drop  of  the  suspension  on  a  slide,  cover 
with  a  cover  sUp,  run  underneath  the  slip  a  drop  of  iodine  solution  and  observe 
the  stained  starch  granules  under  the  microscope.)  Filter  and  apply  a  protein 
color  reaction  (see  page  97)  to  the  filtrate.  It  should  be  positive  indicating 
that  water-soluble  proteins  were  present  in  the  flovu".  Add  fresh  water  to  the 
dough  and  repeat  the  kneading  process.  Continue  this  procedure  with  fresh 
addition  of  water  until  practically  no  starch  granules  are  noted  in  suspension. 
To  a  small  piece  of  the  yellow,  fibrous  gluten  apply  Millon's  Reaction  (page  97). 
This  test  shows  gluten  to  be  protein  material,  Utihze  the  remainder  of  the 
gluten  in  the  preparation  of  gliadin  (page  112). 

Glutenin :  Preparation  and  Tests. — ^(In  the  preparation  of  gliadin  (page  112) 
it  is  customary  to  remove  this  prolamin  from  the  crude  gluten  by  extracting  with 
70  per  cent  alcohol.  Inasmuch  as  gluten  consists  chiefly  of  gliadin  and  glutenin 
the  portion  of  the  gluten  remaining  after  the  extraction  of  the  alcohol-soluble 
protein  gliadin  may  be  utilized  for  the  preparation  of  glutenin.) 

To  the  finely  divided  residue  from  the  preparation  of  gliadin  (page  112)  in  a 
flask  or  bottle  add  about  250  c.c.  of  70  per  cent  alcohol.  Allow  to  stand  for  about 
48  hours  with  repeated  shaking.  This  alcohol  treatment  will  remove  the  gliadin 
and  leave  crude  glutenin.  To  purify  the  glutenin  treat  it  in  a  mortar,  with  suffi- 
cient 0.2  per  cent  NaOH  to  dissolve  it,  and  filter  the  liquid  through  a  wet  pleated 
filter.  NeutraUze  the  filtrate  carefully,  with  0.2  per  cent  HCl  adding  the  acid 
drop  by  drop  with  thorough  mixture  after  each  addition.  (The  glutenin  is  sol- 
uble in  excess  of  acid.)  Filter  off  the  glutenin  precipitate  and  wash  several 
times  with  70  per  cent,  alcohol  and  finally  with  water.     Apply  the  following  tests : 

1.  Solubility  in  water,  salt  solution,  0.2  per  cent  HCl  and  0.5  per  cent  Na2C03. 

2.  Millon's  Reaction. 

Prolamins   (Alcohol-soluble  Proteins) 

The  term  prolamin  has  been  proposed  by  Osborne  for  the  group  of 
proteins  formerly  termed  "alcohol-soluble  proteins."  The  name  is 
very  appropriate  inasmuch  as  these  proteins  yield,  upon  hydrolysis, 
especially  large  amounts  of  proline  and  ammonia.  The  prolamins  are 
simple  proteins  which  are  insoluble  in  water,  absolute  alcohol  and  other 
neutral  solvents,  but  are  soluble  in  70  to  80  per  cent  alcohol  and  in  dilute 
acids  and  alkalis.  They  occur  widely  distributed,  particularly  in  the 
vegetable  kingdom.  The  only  prolamins  yet  described  are  the  zein  of 
maize,  the  hordein  of  barley,  the  gliadin  of  wheat  and  rye,  and  the  hynin 
of  malt.     They  yield  relatively  large  amounts  of  glutamic  acid  on  hy- 

1  This  experiment  as  well  as  those  on  glutenin  and  gliadin  which  follow  have  been 
adapted  from  directions  given  in  Laboratory  Notes  of  Professor  Gies,  College  of  Physicians 
and  Surgeons,  New  York. 


112  PHYSIOLOGICAL    CHEMISTRY 

drolysis  but  no  lysin.  The  largest  percentage  of  glutamic  acid  (43.66 
per  cent)  ever  obtained  as  a  decomposition  product  of  a  protein  sub- 
stance has  very  recently  been  obtained  by  Osborne  and  Guest  from  the 
hydrolysis  of  the  prolamin  gliadin}  This  yield  of  glutamic  acid  is  also 
the  largest  amount  of  any  single  decomposition  product  yet  obtained 
from  any  protein  except  protamines. 

Gliadin :  Preparation  and  Tests. — Introduce  the  finely  divided  crude  gluten 
as  prepared  on  page  1 1 1  into  a  flask  or  bottle,  add  about  250  c.c.  of  70  per  cent 
alcohol-  and  allow  the  mixture  to  stand  24  hoixrs  with  occasional  shaking.  Filter 
(retaining  the  undissolved  portion  for  preparation  of  glutenin,  page  in),  evaporate 
the  filtrate  to  drjmess  in  a  porcelain  dish  over  a  water -bath.  Pulverize  the  dry 
material.    Apply  the  following  tests  to  this  gUadin  powder : 

Solubility  and  Protein  Tests. — Test  the  solubihty  in  alcohol  (30  per  cent, 
50  per  cent  and  70  per  cent),  water,  0.9  per  cent  NaCl,  0.2  per  cent  HCl  and  0.5 
per  cent  Na2C03.  Shake  each  test  repeatedly  and  filter.  To  the  filtrate  apply 
Coagulation  test  (page  105)  and  Biuret  test  (page  98). 

Albuminoids  (Scleroproteins) 

The  albuminoids  yield  similar  hydrolytic  products  to  those  obtained 
from  the  other  simple  proteins  already  considered,  thus  indicating  that 
they  possess  essentially  the  same  chemical  structure.  They  differ  from 
all  other  proteins,  whether  simple,  conjugated,  or  derived,  in  that  they 
are  insoluble  in  all  neutral  solvents.  The  albuminoids  include  "the 
principal  organic  constituents  of  the  skeletal  structure  of  animals  as 
well  as  their  external  covering  and  its  appendages."  Some  of  the  princi- 
pal albuminoids  are  keratin,  elastin,  collagen,  reticulin,  spongin,  and 
fibroin.  Gelatin  cannot  be  classed  as  an  albuminoid  although  it  is  a 
transformation  product  of  collagen.  The  various  albuminoids  differ 
from  each  other  in  certain  fundamental  characteristics  which  will  be 
considered  in  detail  under  Epithelial  and  Connective  Tissue  (sec 
Chapter  XVIII). 

CONJUGATED  PROTEINS 

Conjugated  proteins  consist  of  a  protein  molecule  united  to  some 
other  molecule  or  molecules  otherwise  than  as  a  salt.  We  have  glyco- 
proteins, nude 0 proteins,  hemoglobins  (chromoproteins),  phosphoproteins 
and  lecithoproteins  as  the  five  classes  of  conjugated  proteins. 

Glycoproteins  may  be  considered  as  compounds  of  the  protein  mole- 

1  Osborne  and  Guest:  Jour.  Biol.  Chem.,  9,  425,  1911.  Up  to  this  time  the  yield  of 
41.32  per  cent  obtained  by  Kleinschmitt  from  hordein  was  the  maximum  yield. 

2  Bailey  and  Blish  claim  that  50  per  cent  alcohol  is  more  satisfactory  {Jour.  Biol. 
Chem.,  23,  345,  1915). 


s. 

C. 

H. 

0. 

2-33 

48.76 

6.53 

30.60 

2.32 

47-43 

6.63 

31-40 

PROTEINS  113 

cule  with  a  substance  or  substances  containing  a  carbohydrate  group 
other  than  a  nucleic  acid.  The  glycoproteins  yield,  upon  decomposition, 
protein  and  carbohydrate  derivatives,  notably  glucosamine,  CH2OH.- 
(CHOH)3.CH(NH2).CHO,  and  galactosamine,  OHCHs.fCHOHjg.CH- 
(NH2).CH0.  The  principal  glycoproteins  are  mucoids,  mucins,  and 
chondroprotcins.  By  the  term  mucoid  we  may  in  general  designate 
those  glycoproteins  which  occur  in  tissues,  such  as  tendomucoid  from 
tendinous  tissue  and  osseomucoid  from  bone.  (For  the  preparation  of 
tendomucoid  see  Chapter  XVIII.)  The  elementary  composition  of 
these  typical  mucoids  is  as  follows: 

N. 

Tendomucoid^ ii-75 

Osseomucoid- 12.22 

The  term  mucins  may  be  said  in  general  to  include  those  forms  of  glyco- 
proteins which  occur  in  the  secretions  and  fluids  of  the  body  (For  the 
preparation  of  salivary  mucin  see  Chapter  III.)  Chondroproteins  are 
so  named  because  chondrom>ucoid,  the  principal  member  of  the  group, 
is  derived  from  cartilage  (chondrigen) .  A  myloid,^  which  appears  patho- 
logically in  the  spleen,  liver,  and  kidneys,  is  also  a  chondroprotein. 

The  phospho proteins  are  considered  to  be  "compounds  of  the 
protein  molecule  and  some,  as  yet  undefined,  phosphorus-containing 
substances  other  than  a  nucleic  acid  or  lecithin."  The  percentage  of 
phosphorus  in  phosphoproteins  is  very  similar  to  that  in  nucleoproteins, 
but  they  differ  from  this  latter  class  of  proteins  in  that  they  do  not 
yield  any  purine  bases  upon  hydrolytic  cleavage.  Two  of  the  common 
phosphoproteins  are  the  casein  of  milk  and  the  ovoviteUin  of  the  egg- 
yolk.  The  phosphorus  in  these,  as  in  all  proteins,  exists  in  phosphoric 
acid  radicals.  For  the  preparation  of  a  typical  phosphoprotein  (casein) 
see  Chapter  XVII. 

The  hemoglobins  (chromoproteins)  are  compounds  of  the  protein 
molecule  with  hematin  or  some  similar  substance.  The  principal  mem- 
ber of  the  group  is  the  hemoglobin  of  the  blood.  Upon  hydrolytic  cleav- 
age this  hemoglobin  yields  a  protein  termed  glohin  and  a  coloring  matter 
termed  hcmochromogcn.  The  latter  substance  contains  iron  and  upon 
coming  into  contact  with  oxygen  is  oxidized  to  form  hematin.  Hemo- 
cyanin,  another  member  of  the  class  of  hemoglobins,  occurs  in  the  blood 
of    certain    invertebrates,    notably    ccphalopods,    gastcropods,    and 

'  Chittenden  and  Gies:  Jour.  Exp.  Med.,  i,  1S6,  1S96. 
■^  Hawk  and  Oies:  Anier.  Jour.  Physiol.,  5,  387,  1901. 

^  Not  to  be  confused  \vitl\  tlie  suVjstance  awyloid  which  may  be  formed  from  cellulose 
(see  p.  49). 


114  PHYSIOLOGICAL    CHEMISTRY 

Crustacea.  Hemocyanin  generally  contains  either  copper,  manganese, 
or  zinc  in  place  of  the  iron  of  the  hemoglobin  molecule.  For  the  prepa- 
ration of  hemoglobin  in  crystaUine  form  see  Chapter  XV. 

The  lecithoproteins  consist  of  a  protein  molecule  joined  to  lecithin. 
They  have  been  comparatively  little  studied  and  may  possibly  be 
mixtures  of  protein  and  lecithin. 

For  consideration  of  nucleoproteins  see  Chapter  VL 

DERIVED  PROTEINS 

These  substances  are  derivatives  which  are  formed  through  hydro- 
lytic  changes  of  the  original  protein  molecule.  They  may  be  di\'ided 
into  two  groups,  the  primary  protein  derivatives  and  the  secondary 
protein  derivatives.  The  term  secondary  derivatives  is  made  use  of 
in  this  connection  since  the  formation  of  the  primary  derivatives  gener- 
ally precedes  the  formation  of  these  secondary  derivatives.  These 
derived  proteins  are  obtained  from  native  simple  proteins  by  hy- 
drolyses  of  various  kinds,  e.g..  through  the  action  of  acids,  alkalis, 
heat,  or  enz}Tnes.  The  particular  class  of  derived  protein  desired 
regulates  the  method  of  treatment  to  which  the  native  protein  is 
subjected. 

Primary  Protein  Derivatives 

The  primary  protein  derivatives  are  "apparently  formed  through 
hydrolytic  changes  which  involve  only  sHght  alterations  of  the  protein 
molecule.'"  This  class  includes  proteans,  metaproteins  and  coagulated 
proteins. 

PROTEANS 

Proteans  are  those  insoluble  protein  substances  which  are  produced 
from  proteins  originally  soluble  through  the  incipient  action  of  water, 
enz}Tnes,  or  very  dilute  acids.  It  is  well  known  that  globulins  become 
insoluble  upon  repeated  reprecipitation  and  it  may  possibly  be  found  that 
the  greater  number  of  the  proteans  are  transformed  globulins.  Osborne, 
however,  beUeves  that  nearly  all  proteins  may  give  rise  to  proteans. 
This  investigator  who  has  so  very  thoroughly  investigated  many  of 
the  vegetable  proteins  claims  that  the  hydrogen  ion  is  the  active  agent 
in  the  transformation.  The  protein  produced  from  the  transformation 
of  edestin  is  called  edestan,  that  produced  from  myosin  is  called  myosan, 
etc.  The  name  protean  was  first  given  to  this  class  of  proteins  by  Os- 
borne in  1900  in  connection  with  his  studies  of  edestin. 


PROTEINS  I I 


Experiments  on  Proteans 


Preparation  and  Study  of  Edestan. — Prepare  edestin  according  to  the  direc- 
tions given  on  page  log.  Bring  the  edestin  into  solution  in  0.2  per  cent  hydro- 
chloric acid  and  permit  the  acid  solution  to  stand  for  about  one-half  hour.^  Neutral- 
ize with  a  0.5  per  cent,  solution  of  sodium  carbonate,  filter  off  the  precipitate  of 
edestan  and  make  the  following  tests: 

1.  Solubility. — Try  the  solubility  in  water,  sodium  chloride,  dilute  acid  and 
alkali.  Note  the  altered  solubility  of  the  edestan  as  compared  with  that  of  edestin 
(see  page  109). 

2.  Millon's  Reaction. 

3.  Coagulation  Test. — Place  a  small  amount  of  the  protean  in  a  test-tube, 
add  a  little  water  and  boil.  Now  add  dilute  hydrochloric  acid  and  note  that 
the  protein  no  longer  dissolves.      It  has  been  coagulated. 

4.  Tests  on  Edestan  Solution. — Dissolve  the  remainder  of  the  edestan  pre- 
cipitate in  0.2  per  cent  hydrochloric  acid  and  make  the  following  tests: 

(a)  Biuret  Test. 

(b)  Influence  of  Protein  Precipitants. — Try  a  few  protein  precipitants  such  as 
picric  acid  and  mercuric  chloride. 

METAPROTEINS 

The  metaproteins  are  formed  from  the  native  simple  proteins 
through  an  action  similar  to  that  by  which  proteans  are  formed.  In 
the  case  of  the  metaproteins,  however,  the  changes  in  the  original  pro- 
tein molecule  are  more  profound.  These  derived  proteins  are  char- 
acterized by  being  soluble  in  very  weak  acids  and  alkalis,  but  insoluble 
in  neutral  fluids.  The  metaproteins  were  formerly  termed  albuminates, 
but  inasmuch  as  the  termination  ate  signifies  a  salt  it  has  always  been 
somewhat  of  a  misnomer. 

Two  of  the  principal  metaproteins  are  the  acid  metaprotein  or  so- 
called  acid  albuminate  and  the  alkali  metaprotein  or  so-called  alkali 
albuminate.  They  differ  from  the  native  simple  proteins  principally  in 
being  insoluble  in  sodium  chloride  solution  and  in  not  being  coagulated 
except  when  suspended  in  neutral  fluids.  Both  forms  of  metaprotein 
are  precipitated  upon  the  approximate  neutralization  of  their  solutions. 
They  are  precipitated  by  saturating  their  solutions  with  ammonium  sul- 
phate, and  by  sodium  chloride  also,  provided  they  are  dissolved  in 
an  acid  solution.  Acid  metaprotein  contains  a  higher  percentage  of 
nitrogen  and  sulphur  than  the  alkali  metaprotein  from  the  same  source, 
since  some  of  the  nitrogen  and  sulphur  of  the  original  protein  is  liberated 
in  the  formation  of  the  latter.  Because  of  this  fact,  it  is  impossible 
to  transform  an  alkali  metaprotein  into  an  acid  metaprotein,  while  it 
is  possible  to  reverse  the  process  and  transform  the  acid  metaprotein 
into  the  alkali  modification. 

^  The  edestan  solution  preserved  from  experiment  (5),  p.  no,  may  be  used. 


il6  physiological  chemistry 

Experiments  on  Metaproteins 

AGED  METAPROTEIN  (ACID  ALBUMINATE) 

Preparation  and  Study. — Take  25  grams  of  hashed  lean  beef  washed  free 
from  the  major  portion  of  blood  and  inorganic  matter,  and  place  it  in  a  medium- 
sized  beaker  with  100  c.c.  of  0.2  per  cent  HCl.  Place  it  on  a  boiUng  water-bath 
for  one -half  hour,  filter,  cool,  and  divide  the  filtrate  into  two  parts.  Neutralize 
the  first  part  with  dilute  KOH  solution,  filter  off  the  precipitate  of  acid  metapro- 
tein  and  make  the  following  tests : 

(i)  Solubility. — SolubiUty  in  the  ordinary  solvents  (see  page  21). 

(2)  Millon's  Reaction. 

(3)  Coagulation  Test— Suspend  a  little  of  the  metaprotein  in  water  (neutral 
solution)  and  heat  to  boiUng  for  a  few  moments.  Now  add  1-2  drops  of  KOH 
solution  to  the  water  and  see  if  the  metaprotein  is  still  soluble  in  dilute  alkali. 
What  is  the  result  and  why? 

(4)  Test  for  Unoxidized  Sulphur  (see  page  108). 

Subject  the  second  part  of  the  original  solution  to  the  following  tests : 

(5)  Coagulation  Test. — Heat  some  of  the  solution  to  boiling  in  a  test-tube. 
Does  it  coagulate? 

(6)  Biuret  Test. 

(7)  Influence  of  Protein  Precipitants. — Try  a  few  protein  precipitants  such  as 
picric  acid  and  mercuric  chloride.  How  do  the  results  obtained  compare  with 
those  from  the  experiments  on  egg  albumin?     (See  page  103.) 

ALKALI  METAPROTEIN  (ALKALI  ALBUMINATE) 

Preparation  and  Study. — Carefully  separate  the  white  from  the  yolk  of  a 
hen's  egg  and  place  the  former  in  an  evaporating  dish.  Add  concentrated  potas- 
sium hydroxide  solution,  drop  by  drop,  stirring  continuously.  The  mass  gradu- 
ally thickens  and  finally  assumes  the  consistency  of  jelly.  This  is  solid  alkali 
metaprotein  or  "Lieberkiihn's  jelly."  Do  not  add  an  excess  of  potassium  hydrox- 
ide or  the  jelly  will  dissolve.  Cut  it  into  small  pieces,  place  a  cloth  or  wire  gauze 
over  the  dish,  and  by  means  of  running  water  wash  the  pieces  free  from  adherent 
alkaU.  Now  add  a  small  amount  of  water,  which  forms  a  weak  alkahne  solution 
with  the  alkaU  within  the  pieces,  and  dissolve  the  jelly  by  gentle  heat.  Cool  the 
solution  and  divide  it  into  two  parts.  Proceed  as  follows  with  the  first  part: 
Neutralize  with  dilute  hydrochloric  acid,  noting  the  odor  of  the  Uberated  hydro- 
gen sulphide  as  the  alkaH  metaprotein  precipitates.  Filter  off  the  precipitate 
and  test  as  for  acid  metaprotein  (tests  i,  2,  3  and  4),  above,  noting  particularly 
the  sulphur  test.  How  does  this  test  compare  with  that  given  by  the  acid  meta- 
protein? Make  tests  on  the  second  part  of  the  solution  the  same  as  for  acid 
metaprotein  (tests  5,  6  and  7)  above. 

Coagulated  Proteins 

These  derived  proteins  are  produced  from  unaltered  protein  mate- 
rials by  heat,  by  long  standing  under  alcohol,  or  by  the  continuous 
movement  of  their  solutions  such  as  that  produced  by  rapid  stirring  or 
shaking.     In  particular  instances,  such  as  the  formation  of  fibrin  from 


PROTEINS  I  I  7 

fibrinogen  (see  page  256),  the  coagulation  may  be  produced  by  enzyme 
action.  Ordinary  soluble  proteins  after  having  been  transformed  into 
the  coagulated  modification  are  no  longer  soluble  in  the  ordinary  sol- 
vents. Upon  being  heated  in  the  presence  of  strong  acids  or  alkalis, 
coagulated  proteins  are  converted  into  metaproteins. 

Many  proteins  coagulate  at  an  approximately  fixed  temperature 
under  defim'te  conditions  (see  pages  105  and  339).  This  characteristic 
may  be  applied  to  separate  difierent  coagulable  proteins  from  the  same 
solution  by  fractional  coagulation.  The  coagulation  temperature  fre- 
quently may  serve  in  a  measure  to  identify  proteins  in  a  manner  similar 
to  the  melting-point  or  boiling-point  of  many  other  organic  substances. 
The  separation  of  proteins  by  fractional  coagulation  is  thus  analogous 
to  the  separation  of  volatile  substances  by  means  ol  fractional  distillation. 
This  method  of  separating  proteins  is  not  a  satisfactory  one,  however, 
inasmuch  as  proteins  in  solution  have  difierent  eft'ects  upon  one  another 
and  also  because  of  the  fact  that  the  nature  of  the  solvent  causes  a 
variation  in  the  temperature  at  which  a  given  protein  coagulates.  The 
nature  of  the  process  involved  in  the  coagulation  of  proteins  by  heat 
is  not  well  understood,  but  it  is  probable  that  in  addition  to  the  altered 
arrangement  of  the  component  atoms  in  the  molecule,  there  is  a  mild 
hydrolysis  which  is  accompanied  by  the  liberation  of  minute  amounts 
of  hydrogen,  nitrogen,  and  sulphur.  The  presence  of  a  neutral  salt 
or  a  trace  of  a  mineral  acid  may  facilitate  the  coagulation  of  a  protein 
solution  (see  page  105),  whereas  any  appreciable  amount  of  acid  or 
alkali  will  retard  or  entirely  prevent  such  coagulation. 

It  has  been  shown  that  the  coagulation  of  proteins  by  heat  pro- 
ceeds in  two  stages:^  first,  a  reaction  between  the  protein  and  the  hot 
water  (denaturation),  and  second,  an  agglutination  or  separation  of  the 
altered  protein  in  particulate  form.  The  concentration  of  acid,  or 
hydrogen  ion,  in  the  solution  influences  the  coagulation  of  proteins,  such 
that  the  original  protein  is  acted  upon  less  readily  by  hot  water  alone 
than  in  the  presence  of  acid.  The  formation  of  the  coagulum  is  ac- 
companied by  the  disappearance  of  the  free  acid  from  the  solution, 
indicating  the  formation  of  a  protein  salt.  A  disturbance  of  the  equi- 
librium between  the  hydrolyzed  and  unhydrolyzed  portions  of  the  pro- 
tein salt,  due  to  the  greater  rapidity  with  which  the  unhydrolyzed 
portion  is  precipitated,  results  in  the  gradual  removal  of  both  pro- 
tein and  acid  from  the  solution.  This  has  been  offered  as  an  explana- 
tion of  the  decreasing  acidity. 

According  to  Chick  and  Martin,  the  addition  of  neutral  salts  to  the 
acid  solution  of  the  salt-free  protein  to  be  coagulated  results  in  a  decreased 

^  Chick  and  Martin:  Journal  of  Physiology,  43,  i,  191 1. 


Il8  PHYSIOLOGICAL    CHEMISTRY 

rate  of  coagulation.  This  is  due  in  part  to  the  decrease  in  the  concen- 
tration of  the  free  acid,  which  results  from  the  disturbance  of  the  equilib- 
rium between  the  protein  and  acid  and  also  in  part  to  the  direct  influence 
which  the  salts  exert  upon  the  protein.  The  presence  of  neutral  salts 
may  under  certain  circumstances  facilitate  the  coagulation  of  proteins 
by  heat. 

The  temperature  at  which  egg-white  is  coagulated  causes  a  difference 
in  the  appearance  of  the  coagulum.^  Coagulated  egg-white  which  has 
been  immersed  in  water  at  a  low  temperature  and  then  gradually  heated 
to  the  coagulating  temperature  is  more  translucent  and  has  a  bluish 
color,  whereas  egg-white  which  has  been  immersed  in  water  heated  to  a 
temperature  above  the  coagulating  temperature  is  creamy  white  in 
color.     They  also  possess  different  digestibilities. 

Experiments  on  Coagulated  Protein 

Ordinary  coagxilated  egg-white  may  be  used  in  the  following  tests : 

1.  Solubility. — Try  the  solubiUty  of  small  pieces  of  the  coagulated  protein  in 
each  of  the  ordinary  solvents  (see  page  21). 

2.  Millon's  Reaction. 

3.  Xanthoproteic  Reaction. — Partly  dissolve  a  medium-sized  piece  of  the 
protein  in  concentrated  nitric  acid.  Cool  the  solution  and  add  an  excess  of 
ammonium  hydroxide.  Both  the  protein  solution  and  the  undissolved  protein 
will  be  colored  orange. 

4.  Biuret  Test. — Partly  dissolve  a  mediiun-sized  piece  of  the  protein  in  con- 
centrated potassium  hydroxide  solution.  If  the  proper  dilution  of  copper  sul- 
phate solution  is  now  added  the  white  coagulated  protein,  as  well  as  the  protein 
solution,  will  assume  the  characteristic  purphsh-violet  color. 

5.  Glyoxylic  Acid  Reaction  (HopkLns-Cole). — Conduct  this  test  according  to 
the  modification  given  on  page  107. 

Secondary  Protein  Derivatives 

These  derivatives  result  from  a  more  prof  ound  cleavage  of  the  protein 
molecule  than  that  which  occurs  in  the  formation  of  the  primary  deriva- 
tives.    The  class  includes  proteoses,  peptones,  and  peptides. 

PROTEOSES  AND  PEPTONES 

Proteoses  are  intermediate  products  in  the  digestion  of  proteins  by 
proteolytic  enzymes,  as  well  as  in  the  decomposition  of  proteins  by  hy- 
drolysis and  the  putrefaction  of  proteins  through  the  action  of  bacteria. 
Proteoses  are  called  albumoses  by  some  writers,  but  it  seems  more  logical 
to  reserve  the  term  albumose  for  the  proteose  of  albumin. 

Peptones  are  formed  after  the  proteoses  and  it  has  been  customary  to 

*  Frank:  Journal  of  Biological  Chemistry,  9,  463,  191 1. 


PROTEINS  119 

consider  them  as  the  last  product  of  the  processes  before  mentioned 
which  still  possess  true  protein  characteristics.  In  other  words,  ithas 
been  considered  that  the  protein  nature  of  the  end-products  of  the 
cleavage  of  the  protein  molecule  ceased  with  the  peptones,  and  that  the 
simpler  bodies  formed  from  peptones  were  substances  of  a  different 
nature  (see  page  65).  However,  as  the  end-products  have  been  more 
carefully  studied,  it  has  been  found  to  be  no  easy  matter  to  designate 
the  exact  character  of  a  peptone  or  to  indicate  the  exact  point  at 
which  the  peptone  characteristic  ends  and  the  peptide  characteristic 
begins.  The  situation  regarding  the  proteoses,  peptones  and  peptides 
is  at  present  a  most  unsatisfactory  one  because  of  the  unsettled  state 
of  our  knowledge  regarding  them.  The  exact  differences  between 
certain  members  of  the  peptone  and  peptide  groups  remain  to  be  more 
accurately  established.  It  has  been  quite  well  established  that  the 
peptones  are  peptides  or  mixtures  of  peptides,  but  the  term  peptide  is 
used  at  present  to  designate  only  those  possessing  a  definite  structure. 
There  are  several  proteoses  (protoproteose,  heteroproteose  and 
deuteroproteose),  and  at  least  two  peptones  (amphopeptone  and  anti- 
peptone),  which  result  from  proteolysis.  The  differentiation  of  the 
various  proteoses  and  peptones  at  present  in  use  is  rather  unsatisfactory. 
These  compounds  are  classified  according  to  their  varying  solubilities, 
especially  in  ammonium  sulphate  solutions  of  diff"erent  strengths.  The 
exact  differences  in  composition  between  the  various  members  of  the 
group  remain  to  be  more  accurately  established.  Because  of  the 
diflSculty  attending  the  separation  of  these  bodies,  pure  proteose  and 
peptone  are  not  easy  to  procure.  The  so-called  peptones  sold  com- 
mercially contain  a  large  amount  of  proteose.  As  a  class  the  proteoses 
and  peptones  are  very  soluble,  dift'usible  bodies  which  are  non-coagu- 
lable  by  heat.  Peptones  differ  from  proteoses  in  being  more  diffusible, 
non-precipitable  by  (NH4)2S04,  and  by  their  failure  to  give  any  reaction 
with  potassium  ferrocyanide  and  acetic  acid,  potassio-mercuric  iodide 
and  HCl,  picric  acid,  and  trichloracetic  acid.  Peptones  may  be  pre- 
cipitated by  phosphotungstic  acid,  phosphomolybdic  acid,  absolute 
alcohol  and  tannic  acid,  but  an  excess  of  the  precipitant  may  dissolve 
the  precipitate.  The  so-called  primary  proteoses  are  precipitated  by 
HNO3  and  are  the  only  members  of  the  proteose-peptone  group  which 
are  so  precipitated. 

Some  of  the  more  general  characteristics  of  the  proteose-peptone  group  may 
be  noted  by  making  the  following  simple  tests  on  a  proteose-peptone  powder: 
(i )  Solubility. — SolubiUty  in  hot  and  cold  water  and  sodium  chloride  solution. 
(2)  Millon's     Reaction. 
Dissolve  a  little  of  the  powder  in  water  and  test  the  solution  as  follows : 


I20  PHYSIOLOGICAL   CHEMISTRY 

(i)  Precipitation  by  Picric  Acid. — To  5  c.c.  of  proteose -peptone  solution  in  a 
test-tube  add  picric  acid  until  a  permanent  precipitate  forms.  The  precipitate 
disappears  on  heating  and  returns  on  cooling. 

(2)  Precipitation  by  a  Mineral  Acid. — Try  the  precipitation  by  nitric  acid. 

(3)  Coagxilation  Test. — Heat  a  littie  proteose-peptone  solution  to  boiling. 
Does  it  coagulate  Uke  the  other  simple  proteins  studied? 

SEPARATION  OF  PROTEOSES  AND  PEPTONES^ 

Place  50  c.c.  of  proteose-peptone  solution  in  an  evaporating  dish  or  casserole, 
and  half-saturate  it  with  ammoniimi  sulphate  solution,  which  may  be  accom- 
plished by  adding  an  equal  volvune  of  saturated  ammonivun  sulphate  solution. 
At  this  point  note  the  appearance  of  a  precipitate  of  the  primary  proteoses 
(protoproteose  and  hetero-proteose).  Now  heat  the  half -saturated  solution  and 
its  suspended  precipitate  to  boihng  and  saturate  the  solution  with  solid  am- 
monium sulphate.  At  full  saturation  the  secondary  proteoses  (deuteroproteoses) 
are  precipitated.     The  peptones  remain  in  solution. 

Proceed  as  follows  with  the  precipitate  of  proteoses:  Collect  the  sticky 
precipitate  on  a  rubber-tipped  stirring  rod  or  remove  it  by  means  of  a  watch 
glass  to  a  small  evaporating  dish  and  dissolve  it  in  a  littie  water.  To  remove  the 
ammonium  sulphate,  which  adhered  to  the  precipitate  and  is  now  in  solution, 
add  barium  carbonate,  boil,  and  filter  off  the  precipitate  of  barium  sulphate. 
Concentrate  the  proteose  solution  to  a  small  volume ^  and  make  the  follow- 
ing tests : 

(i)  Biuret   Test. 

(2)  Precipitation  by  Nitric  Acid. — ^What  would  a  precipitate  at  this  point 
indicate? 

(3)  Precipitation  by  Trichloracetic  Acid. — This  precipitate  dissolves  on  heating 
and  returns  on  cooling. 

(4)  Precipitation  by  Picric  Acid. — This  precipitate  also  disappears  on  heat- 
ing and  returns  on  cooling. 

(5)  Precipitation  by  Potassio-mercuric  Iodide  and  Hydrochloric  Acid. 

(6)  Coagulation  Test. — Boil  a  littie  in  a  test-tube.    Does  it  coagulate? 

(7)  Acetic  Acid  and  Potassium  Ferrocyanide  Test. 

The  solution  containing  the  peptones  should  be  cooled  and  filtered,  and  the 
ammonium  sulphate  in  solution  removed  by  boiling  with  barium  carbonate  as 
described  above.  After  filtering  off  the  barium  sulphate  precipitate,  concentrate 
the  peptone  filtrate  to  a  small  volume  and  repeat  the  tests  as  given  under  the 
proteose  solution,  above.  Also  try  the  precipitation  by  phosphotungstic  acid 
and  by  tannic  acid.  In  the  biuret  test  the  solution  should  be  made  very  strongly 
alkaUne  with  solid  potassium  hydroxide. 

PEPTIDES 

The  peptides  are  "definitely  characterized  combinations  of  two  or 
more  amino  acids,  the  carboxyl  (COOH)  group  of  one  being  united 

^  The  separation  of  proteoses  and  peptones  by  means  of  fractional  precipitation  with, 
ammonium  sulphate  does  not  possess  the  significance  it  was  once  supposed  to  possess  inas- 
much as  the  boundary  between  these  substances  and  peptides  is  not  well  defined  (see  p.  119). 

^  If  the  proteoses  are  desired  in  powder  form,  this  concentrated  proteose  solution  may 
now  be  precipitated  by  alcohol,  and  this  precipitate,  after  being  washed  with  absolute 
alcohol  and  with  ether,  may  be  dried  and  powdered. 


PROTEINS 


with  the  amino  (NH2)  group  of  the  other  with  the  elimination  of  a  mole- 
cule of  water."  These  peptides  are  more  fully  discussed  on  pages  70 
and  119. 

REVIEW  OF  PROTEINS 

In  order  to  facilitate  the  student's  review  of  the  proteins,  the  prepara- 
tion of  a  chart  similar  to  the  model  given  is  recommended.  The  signs  -4- 
and  —  may  be  conveniently  used  to  indicate  positive  and  negative 
reactions. 

MODEL  CHART  FOR  REVIEW  PURPOSES 


Solubility 

s 

"3 
O 
e 
'S 

S 

Salting- 
Precipitation  Tests                 out 

Tests 

- 

Protein 
f 

Water 
10%  NaCl 

O 

6 
u 
5 

6 

Cone.  HCl 
Cone.  KOH 

o* 

z 
w 

•d 

2 
a 

Metallic  Salt  (HgCh) 

Alcohol 

Pot.  Perrocyanide 
+  Acetic  Acid 

Potassio-mercuric 
Iodide -f  HCl 

Picric  Acid 
Trichloracetic  Acid 

(NH4)2S04 

0 

2 

Diffusion 
Coagulation  by  1 

Albumin 
Globulin 

--• — 





- 

Nucleoprotein 

1         :     , 

Phosphoprotein 

1 

i 

Glucoprotein 

Acid  metaprotein 

Alkali  metaprotein 

i  Proteose 

J_ 

1  Peptone                            i        I 

• 

;  Coagulated  protein  |        i        1 

1 

"Unknown"  Mixtures  and  Solutions  of  Proteins 

At  this  point  the  student's  knowledge  of  the  characteristics  of  the 
various  proteins  studied  will  be  tested  by  requiring  him  to  examine  several 
"unknown"  protein  mixtures  or  solutions  and  make  full  report  upon  the 
same.    The  scheme  given  on  page  122  may  be  used  in  this  examination. 


122 


PHYSIOLOGICAL    CHEMISTRY 


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CHAPTER  VI 
NUCLEIC  ACIDS  AND  NUCLEOPROTEINS^ 

The  Nucleoproteins. — The  nucleoproteins  occur  widely  distributed 
in  the  animal  and  plant  kingdoms,  being  found  in  nearly  all  cells  and 
particularly  in  the  nuclei  of  cells.  They  are  found  in  especially  large 
amounts  in  glandular  tissues  such  as  those  of  the  thymus,  pancreas  and 
spleen.  The  nucleoproteins  are  combinations  of  protein  with  a  phos- 
phorus-containing substance  known  as  nucleic  acid.  As  different  nu- 
cleic acids  exist  and  are  found  in  combination  with  different  proteins, 
a  variety  of  nucleoproteins  exist.  The  protein  combined  with  the 
nucleic  acid  is  in  certain  cases  a  histone,  the  conjugated  protein  in 
this  case  being  called  a  nucleohistone. 

The  nucleoproteins  give  the  ordinary  protein  color  reactions. 
They  are  acidic  in  character  and  insoluble  in  water.  They  are  readily 
soluble  in  weak  alkali  but  are  precipitated  from  such  solution  on  the 
addition  of  acetic  acid  in  excess  of  which  they  dissolve  with  more  or 
less  difficulty  although  readily  soluble  in  very  dilute  hydrochloric  acid. 
We  distinguish  them  from  mucins,  which  are  likewise  precipitated  by 
acetic  acid  through  the  fact  that  the  latter  give  no  tests  for  phosphorus 
on  decomposition. 

The  nucleoproteins  are  very  complex  and  unstable  substances 
and  one  has  probably  never  been  prepared  in  a  pure  form.  Under  the 
action  of  the  gastric  juice  or  of  weak  acid  nucleoproteins  lose  a  portion 
of  their  protein  content  and  are  transformed  into  a  rather  ill-defined 
class  of  substances  known  as  micleins  w'hich  still  possess  some  protein 
in  combination  with  the  nucleic  acid  molecule.  In  most  cases  the 
decomposition  does  not  proceed  further  in  gastric  digestion.  Through 
the  action  of  the  pancreatic  juice,  however,  the  remainder  of  the 
protein  is  split  off  and  the  nucleic  acid  set  free.  The  decomposition 
of  nucleoprotein  may  be  diagramatically  expressed  thus,  although 
the  course  of  decomposition  is  probably  not  quite  so  simple  as  indicated. 

^  For  review  of  the  literature  on  nucleic  acids  and  nucleases  see  Monograph  on  "Nucleic 
Acids"  by  Walter  Jones,  New  York,  19 14,  Longmans  Green  &  Co. 

123J 


124  PHYSIOLOGICAL    CHEMISTRY 

NTJCLEOPROTEIN 

.  I. 

(gastric  digestion) 


Protein  Nuclein 

I 

(pancreatic  digestion) 


Protein  Nucleic  Acid 

The  Nucleic  Acids. — The  nucleic  acids  of  the  animal  body  occur 
mainly  in  combination  with  protein  material  in  the  so-called  nucleo- 
proteins  of  which  they  form  the  characteristic  radicals  (see  page  123). 
The  amount  and  character  of  the  protein  with  which  the  nucleic  acid 
molecule  is  combined  varies  and  the  acid  may  in  certain  cases  be  found 
in  cells  in  a  free  form.  Naturally  those  tissues  are  richest  in  nucleic 
acid  which  contain  the  largest  amount  of  nuclear  material  and  of 
nucleoprotein.  Such  are  the  glandular  tissues  of  the  body  as  the 
thymus,  spleen,  pancreas,  liver,  etc.  The  heads  of  the  spermatozoa 
consist  almost  entirely  of  nucleic  acid  in  combination  with  protamine. 

The  nucleic  acids  are  a  distinct  class  of  substances,  characterized 
by  their  decomposition  products.  They  are  strongly  acid  in  reaction 
and  contain  considerable  phosphorus.  They  may  be  divided  into  two 
main  groups,  the  animal  and  the  plant  nucleic  acids.  The  two 
classes  differ  in  certain  respects  but  all  of  the  true  animal  nucleic  acids 
appear  to  be  practically  identical  in  composition.  Animal  nucleic 
acid  is  most  readily  prepared  from  the  thymus  while  plant  nucleic 
acid  is  most  readily  obtained  from  yeast. 

The  nucleic  acids  are  difficultly  soluble  in  cold  water,  more  readily 
in  hot  water,  insoluble  in  alcohol,  but  readily  soluble  in  weak  alkali 
with  the  formation  of  the  alkali  salt.  If  pure  they  do  not  give  the 
protein  color  reactions.  They  are  optically  active.  They  are  pre- 
cipitated from  their  alkaline  solutions  by  HCl,  but  only  the  plant  nu- 
cleic acid  is  precipitated  by  acetic  acid.  In  weak  acid  solution  they  are 
precipitated  by  protein  the  combination  being  considered  a  "nuclein." 
They  form  insoluble  salts  with  alkaline  earth  and  heavy  metals. 
The  sodium  salt  of  animal  nucleic  acid  in  4  per  cent  solution  is  liquid 
while  warm  but  solidifies  to  a  gelatinous  mass  on  cooling.  Plant 
nucleic  acid  does  not  do  this. 

The  nucleic  acids  on  hydrolysis  yield  phosphoric  acid,  purine  and 
pyrimidine  bases,  and  a  carbohydrate  or  carbohydrate  derivative.  The 
composition  varies  slightly  with  the  type  of  nucleic  acid.  Plant  nucleic 
acids  contain  a  pentose  group  while  animal  nucleic  acids  contain  a 


NUCLEIC   ACIDS    AND    NUCLEOPROTEINS  1 25 

hexose  group.  Both  types  contain  the  purine  bases,  guanine  and 
adenine  and  the  pyrimidine  base  cytosine.  Plant  nucleic  acid  contains 
also  the  pyrimidine  base  uracil,  which  in  the  animal  nucleic  acid  is  sub- 
stituted by  the  base  thymine.  The  nucleic  acids  are  not,  however, 
simple  substances  whose  molecules  contain  a  single  phosphoric  acid  or 
carbohydrate  group.  They  are  apparently  combinations  of  several 
radicals  known  as  nucleotides  each  of  which  contains  one  car- 
bohydrate group  combined  with  a  single  base  and  a  single  phosphoric 
acid  molecule.  Thus  the  following  structural  formula  has  been  given 
to  yeast  nucleic  acid  by  Levene  and  Jacobs^  indicating  that  it  contains 
four  nucleotide  radicals  and  may  hence  be  called  a  tetranucleotide. 

HO 

\ 

0  =  POC5H803C5H4N50 

/  Guanine  group 

o 

\ 

0-POC5H803C5H4N5 

/  Adenine  group 

0 

\ 

0=POC5H803C4H3N202 

/  Uracil  group 

o 

\ 

0  =  POC5H803C4H4N30 

/  Cytosine  group 

HO 

Yeast  nucleic  acid  (tetranucleotide) 

The  cleavage  of  the  nucleic  acid  molecule  into  its  corresponding 
nucleotides  is  brought  about  during  digestion  by  enzymes  present  in 
the  intestinal  juice  and  intestinal  mucosa.  Enzymes  of  similar  origin 
act  further  on  the  nucleotides  thus  formed  and  split  off  the  phosphoric 
acid  radicals  together  with  carbohydrate-base  compounds  which  are 
called  nucleosides.  The  decomposition  prior  to  absorption  does 
not  probably  proceed  further  than  to  the  formation  of  nucleotides 
and  nucleosides.  ]\Iany  tissues  however  contain  enzymes  capable  of 
completing  the  decomposition  with  liberation  of  the  carbohydrate 
and  basic  radicals.  The  purine  bases  may  also  be  deaminized  while 
still  in  combination  as  nucleosides  and  further  hydrolysis  would  then 
lead  to  the  direct  liberation  of  the  oxypurines  instead  of  their  precursors, 
the  amino-purines. 

^Levene  and  Jacobs:  Bcr.  d.  dcitlsch.  Clicm.  Gcs.,  43,  3151.  iQio;  44,  1027,  loii 


126  PHYSIOLOGICAL    CHEMISTRY 

Jones^  has  suggested  a  method  by  which  the  course  of  the  decom- 
position of  the  nucleic  acid  molecule  can  be  followed.  By  this  means 
it  is  readily  shown  that  phosphoric  acid  is  liberated  at  very  different 
rates  from  the  different  nucleotides. 

The  following  outline  will  indicate  the  course  of  decomposition  of/a 
nucleic  acid  and  the  enzymes  involved  in  the  process. 

DECOMPOSITION  OF  NUCLEIC  ACID 
NUCLEIC  ACID 

(nucleicacidase  of  intestinal  mucosa  and  juice) 


I  I 

Purine  Nucleotides  Pyrimidine  Nucleotides 

I  .  I 

(nucleotidase  of  intestinal  (tissue  nucleases) 

mucosa  and  juice)  I 


Phosphoric  Acid  Purine  Nucleosides  55^^^?!,°"^  4"*^ 

I  Pjrrimidme  Bases 

I  Cytosine  and  Thjrmine 

(nucleosidase  of  tissues)  or  Uracil 


Sugar  Piu-ine  Bases 

(pentose  or  hexose)  Adenine 

Guanine 

With  regard  to  the  fate  of  the  various  radicals  of  the  nucleic  acids 
in  the  body  a;fter  absorption  little  is  definitely  known.  The  phosphoric 
acid  may  of  course  be  built  up  into  phosphorus-containing  cell  con- 
stituents such  as  nucleoproteins,  phosphoproteins  or  phosphatides,  or 
be  eliminated  as  phosphate  in  the  urine.  The  carbohydrate  portion 
may  undergo  the  usual  transformations  of  intermediary  carbohydrate 
metabolism.  The  nucleosides  appear  to  be  ordinarily  absorbed  un- 
changed from  the  intestine  and  may  be  to  a  certain  extent  directly  re- 
synthesized  in  the  animal  body  to  nucleoprotein.  The  excess  over  body 
requirement  must,  however,  be  decomposed,  although  a  certain  portion 
may  possibly  be  stored  up  in  the  individual  cells  or  in  certain  organs. 
Enzymes  capable  of  decomposing  nucleic  acids  are  found  in  most  of  the 
cells  of  the  body. 

The  Purine  Bases. — As  has  been  indicated  the  basic  substances 
present  in  nucleic  acid  belong  to  two  classes  the  purine  and  pyrimi- 
dine bases.  The  purine  bases  set  free  on  the  decomposition  of  nucleic 
acid  are  adenine  and  guanine  belonging  to  the  class  of  amino  purines. 

^  Jones:  Presidential  address  before  the  Society  of  Biological  Chemists,  Boston,  Dec. 
27,  iQiS- 


NUCLEIC   ACIDS   AND   NUCLEOPROTEINS 


127 


The  fate  of  the  amino  purines  in  the  animal  body  is  of  considerable 
interest.  It  has  been  shown  that  certain  tissues  contain  enzymes 
which  transform  these  amino  purines  first  to  corresponding  oxypurines 
known  as  hypoxanthine  and  xanthine  and  finally  to  uric  acid.  It  is 
probable  that  different  enzymes  enter  into  the  various  steps  of  these 
transformations  leading  to  the  formation  of  uric  acid.  Still  another 
enzyme  carries  the  oxidation  further  with  the  formation  of  the  com- 
pound allantoin.  This  enzyme  is  known  as  uricase.  The  [purine 
enzymes  are  widely  distributed  in  tissues.  The  transformations 
brought  about  are  indicated  in  the  following  diagrams. 


N=CNH2 


HN— CO 


HC     C— NH      +H2O— NHa-^    HC     C— NH 


Adenase 


// 


CH 


N— C— N 

Adenine 
6-aniino  purine 


N— C— N' 

Hypoxan'.hxnt 
6-oxypurine 


/ 


CH 


+0 


Hypoxanthine 
oxidase 


HN— CO 

r  I 

H2NC     C— NH 


HN— CO 

I       I 
OC     C— NH 


CH;+H20  —  NH3->  CH 

//  Ciianase  /^ 

N— C— N  HN— C— N 


Guanine 
2-amino-6-oxypurine 


Xanthine 
2-6-dioxypurine 


+0 


Xanthine 
oxidase 


NH2 


CO     CO— NH  O 

I  I  \  Uricase 

CO 


HN— CO 

I       I 
OC     C— NH 


CO 


NH— CH— NH 

Allantcin 


HN— C— NH 

Uric  acid 
2-6-8-trioxypurine 


128  PHYSIOLOGICAL    CHEMISTRY 

All  of  the  physiologically  important  purine  bodies  are  precipitated 
by  ammoniacal  silver  nitrate  solution  in  the  cold  and  by  copper  sul- 
phate and  sodium  bisulphite  in  boiling  solutions.  Some  of  them  are 
readily  identified  by  their  crystalline  forms  or  the  crystalline  forms  of 
certain  of  their  salts.  Uric  acid  differs  from  the  other  purines  in  being 
insoluble  in  dilute  sulphuric  acid.  The  purine  bodies  may  be  distin- 
guished to  a  certain  extent  also  by  the  reactions  which  they  give  when 
their  solutions  are  evaporated  with  nitric  acid  and  the  residue  treated 
with  ammonia.  Uric  acid  gives  the  characteristic  formation  of  the 
purple  murexide  (ammonium  purpurate) .  Potassium  hydroxide  changes 
this  to  a  bluish-violet  color  which  disappears  on  heating.  Xanthine 
and  guanine  form  yellow  compounds  with  nitric  acid  which  turn 
purple  or  violet  on  treating  with  potassium  hydroxide.  The  color  in 
this  case  is  not  lost  by  heating.  Adenine  and  hypoxanthine  do  not 
give  a  color  reaction  with  nitric  acid. 

The  Pyrimidine  Bases. — The  pyrimidine  bases  entering  into  the 
composition  of  nucleic  acid  are  thymine,  cytosine  and  uracil.  Cytosine 
is  found  in  both  types  of  nucleic  acid,  while  thymine  is  found  only 
in  animal  nucleic  acid  and  uracil  only  in  plant  nucleic  acid.  They 
possess  the  following  formulas. 

NH— C=0  NH— C=0  N=C— NH2 

O-C        CH  0=C        C— CH3        0=C        CH 

NH— CH  NH— CH  NH— CH 

Uracil  Thymine  Cytosine 

2-6-dioxypyrimidine  5-methyl-  6-amino- 

2-6-dioxypyrimidine  2-dioxy  pyrimidine 

With  regard  to  the  fate  of  pyrimidine  bases  in  metabolism  very 
little  is  known.  When  the  bases  as  such  are  fed  they  reappear  un- 
changed in  the  urine.  ^  If  nucleic  acid  is  fed  this  does  not  occur  which 
indicates  that  the  pyrimidine  bases  may  undergo  certain  alterations 
in  the  animal  body  while  still  existing  in  combination. 

Experiments 

I.  Preparation  of  Nucleoprotein  from  Yeast. ^ — Place  two  small  cakes  of  ordi- 
nary compressed  yeast  in  a  mortar.  Sprinkle  a  small  horn-spoonful  of  sand  over 
the  yeast,  add  5  c.c.  of  ether  and  10  c.c.  of  water  and  thoroughly  triturate  the 
mixture,  grinding  vigorously.  The  ether  kills  the  yeast,  in  which  condition  the 
comminution  of  the  cells  with  sand  is  more  thoroughly  affected.  Occasionally 
during  the  trituration  process  add  i  or  2  c.c.  of  water  until  the  mixture  is 
comparatively  fluid.     The  whole  process  of  maceration  can  be  completed  in  five 

1  Mendel  and  Myers:  Am.  J.  Physiol.,  26,  77,  1910. 

^  All  experiments  on  nucleoprotein  of  yeast  have  been  taken  from  Laboratory  Notes  of 
Professor  W.  J.  Gies,  of  College  of  Physicians  and  Surgeons,  New  York. 


NUCLEIC   ACIDS    AND    XUCLEOPROTEINS  1 29 

minutes.  Pour  the  thick  liquid  into  a  bottle  aiding  the  transfer  with  enough 
0.4  per  cent  NaOH  to  make  a  final  volume  of  about  125  c.c.  The  alkaU  extracts 
the  nucleoprotein  along  with  the  water-soluble  proteins  of  the  yeast.  Add  a  little 
toluol  and  allow  to  stand  with  frequent  shaking  for  1224  hours.  Filter  through 
a  wet,  fluted  filter.  While  thoroughly  stirring  add  i  drop  at  a  time  of  10  per  cent 
HCl  cautiously  continuing  the  addition  as  long  as  the  milkiness  of  the  mixture  can 
be  increased.  Continue  until  the  protein  completely  separates  and  the  Uquid  is 
practically  clear.  Note  that  the  solution  is  now  acid  in  reaction.  Excess  of  acid 
causes  resolution.  Filter  on  a  wet,  fluted  filter.  Retain  the  precipitate  on  the 
filter  for  nucleoprotein  tests. 

2.  Tests  on  Nucleoprotein. — Try  the  following  tests  on  the  nucleoprotein 
prepared  as  above. 

(a)  Try  the  xanthoproteic  and  Millon's  tests. 

(b)  Test  the  solubiUty  in  water,  10  per  cent  NaCl,  10  per  cent  HCl,  dilute 
KOH,  and  alcohol. 

(c)  Test  for  organically  combined  phosphorus  by  one  of  the  following 
methods. 

Tests  for  Phosphorus  in  Organic  Matter. — i.  Fusion  Test. — To  a  small 
amount  of  the  substance  in  a  crucible  add  about  five  times  its  bulk  of  fusion  mix- 
ture (2  parts  of  sodiiun  carbonate  to  i  of  potassium  nitrate).  Heat  carefully  until 
the  resulting  mixture  is  colorless.  Cool,  dissolve  the  mass  in  a  little  warm  water, 
acidify  with  nitric  acid,  heat  nearly  to  boiling  and  add  a  few  cubic  centimeters  of 
molybdate  solution.  In  the  presence  of  phosphorus  a  yellow  precipitate  of  phos- 
phomolybdate  is  formed. 

Instead  of  acidifying  with  nitric  acid,  the  aqueous  solution  may  be  approxi- 
mately neutralized  with  hydrochloric  acid,  a  few  cubic  centimeters  of  magnesia 
mixture  added  and  then  excess  of  ammonium  hydroxide  solution.  A  white  pre- 
cipitate of  magnesium  ammoniiun  phosphate  is  formed. 

2.  Moist  Ashing  Procedure. — Treat  a  small  amount  of  the  substance  in  a 
large  test-tube  with  about  i  c.c.  of  concentrated  sulphuric  acid.  Then  add  drop  by 
drop  an  equal  volume  of  concentrated  nitric  acid,  and  warm  gently  until  a  clear 
solution  is  obtained.  A  few  more  drops  of  nitric  acid  may  be  added  if  necessary. 
This  treatment  with  sulphuric  and  nitric  acids  must  be  carried  out  with  the 
greatest  caution  particiilarly  when  fatty  substances  are  present  ;  otherwise  an 
explosive  reaction  may  take  place.  Dilute  the  acid  solution  with  a  Uttle  water, 
make  sUghtly  alkaline  with  ammonia  and  then  acid  with  nitric  acid.  Add 
molybdate  solution  and  warm.     A  yellow  precipitate  is  formed. 

(d)  Dissolve  a  little  of  the  precipitate  in  very  dilute  KOH  and  then  make 
slightly  acid  with  acetic  acid. 

(e)  Mix  a  small  portion  of  the  nucleoprotein  with  10  c.c.  of  alcohol.  Filter 
and  wash  free  from  HCl  with  more  alcohol.  (Freedom  from  HCl  is  indicated  by 
absence  of  AgNOa-chloride  reaction  in  the  filtrate.)  Wash  free  from  alcohol 
with  a  httle  water.  Transfer  small  particles  of  the  precipitate  to  moistened  red 
and  blue  litmus  paper  on  a  microscopic  slide.  What  is  the  reaction  of  nucleo- 
protein thus  freed  from  adherent  acid? 

3.  To  Show  the  Presence  of  Purine  Base  Radicals  in  Nucleoprotein.  The 
nucleic  acid  portion  of  the  protein  molecule  contains  phosphoric  acid,  carbohy- 
drate, and  purin  base  radicals  (see  page  126).  Hence  on  the  complete  acid  hydro- 
lysis of  nucleoprotein  material  these  substances  will  be  Uberated  as  well  as  the 
decomposition  products  of  the  protein  part  of  the  molecule.     To  show  their  pres- 

9 


130  PHYSIOLOGICAL    CHEMISTRY 

ence  proceed  as  follows:  Transfer  the  precipitate  of  nucleoprotein  remaining 
from  the  previous  experiment  to  a  small  flask  and  add  25-50  c.c.  of  5  per  cent 
H2SO4.  Boil  for  an  hour  or  more  to  decompose.  Maintain  the  original  volume 
by  adding  water.  The  solution  becomes  brown  due  to  formation  of  melanin- 
like substances.  The  purine  bases  are  set  free.  Retain  one-fourth  of  the  solu- 
tion for  the  next  experiment.  Transfer  the  remainder  to  a  casserole  and  add 
anmionia  with  thorough  mixing,  a  Uttle  at  a  time,  until  the  fimd  is  nearly  neutral. 
Then  make  sUghtly  alkahne  with  dilute  ammonia  and  filter  if  not  clear.  Transfer 
to  a  beaker  and  add  about  10  c.c.  of  5  per  cent  ammoniacal  silver  nitrate  solution. 
Purine  bases  if  present  will  yield  a  brown  fiocculent  precipitate  of  their  silver 
compounds.  If  a  precipitate  does  not  appear  immediately,  examine  the  solution 
after  it  has  been  allowed  to  stand  for  some  time  undisturbed. 

4.  To  Show  the  Presence  of  Protein,  Carbohydrate,  and  Phosphoric  Acid 
Radicals  in  Nucleoprotein.— Filter  the  greater  portion  of  the  acid  liquid  which  was 
reserved  from  the  preceding  experiment.  Apply  the  following  tests  to  portions  of 
it:  (a)  The  biuret  test,  (b)  The  xanthoproteic  test,  (c)  Molisch  test,  (d) 
Fehling's  test,     (e)  Test  for  phosphate. 

5.  Preparation  of  Thymus  Nucleoprotein. — About  100  grams  of  fresh  thymus 
gland  (lymphatic  glands  may  also  be  used)  freed  as  nearly  as  possible  from  adherent 
fat  are  run  through  a  meat  chopper.  To  this  material  in  a  flask  add  300  c.c.  of  0.9 
per  cent  NaCl  and  allow  to  stand  24-48  hours  in  the  cold.  A  little  chloroform  and 
toluol  should  be  added  as  preservatives,  and  the  mixture  shaken  occasionally  during 
this  period.  Filter.  A  milk  white  liquid  is  obtained.  Precipitate  the  nucleo- 
protein from  solution  by  the  careful  addition  of  dilute  acetic  acid.  Excess  of  the 
acid  should  be  avoided.  Ordinarily  acetic  acid  to  make  a  i  per  cent  solution  is 
sufiicient.  Filter  off  the  precipitate.  Wash  with  alcohol  and  then  with  ether  and 
dry. 

6.  Experiments  on  Thymus  Nucleoprotein. — Repeat  the  experiments  given 
under  Yeast  Nucleoprotein  (page  129). 

7.  Preparation  of  Yeast  Nucleic  Acid. — Dilute  50  c.c.  of  i  per  cent  NaOH 
with  250  c.c.  of  water  in  a  casserole  and  add  to  this  solution  100  grams  of  com- 
pressed yeast  cut  in  small  pieces.  Heat  on  the  water-bath  for  half  an  hour  with 
occasional  stirring.  Remove  from  the  bath  and  filter  at  once  through  a  folded 
filter.  To  the  cooled  filtrate  add  acetic  acid  until  faintly  acid  to  Utmus.  Filter 
again.  Evaporate  the  solution  to  100  c.c.  or  less  and  filter  if  necessary.  Allow 
to  cool  to  40°C.  or  below,  then  pour  with  vigorous  stirring  into  200  c.c.  of  95  per 
cent  alcohol  containing  2  c.c.  of  concentrated  HCl.  Allow  to  settle  and  wash  the 
precipitate  by  decantation  in  a  tall  vessel,  twice  with  95  per  cent,  alcohol  and 
twice  with  ether.  Transfer  to  a  filter  paper.  Allow  to  drain  and  dry  at  room 
temperature. 

8.  Tests  on  Nucleic  Acid  from  Yeast.' — i.  Test  the  solubility  of  nucleic  acid 
in  cold  and  hot  water,  in  alcohol,  and  in  dilute  acid  and  alkali.  To  the  solution  in 
alkali  add  dilute  HCl  drop  by  drop  until  the  solution  is  acid,  then  add  excess  of 
concentrated  HCl. 

Does  nucleic  acid  coagulate  on  boiUng?  Does  the  solution  in  hot  water 
gelatinize    on   cooling? 

2.  Try  xanthoproteic  reaction  and  biuret  test. 

3.  Dissolve  a  little  nucleic  acid  in  water  with  the  aid  of  heat.     Test  the  re- 

1  A  satisfactory  preparation  of  yeast  nucleic  acid  may  be  obtained  from  Merck  and  Co. 


NUCLEIC   ACIDS    AND    NUCLEOPROTEINS  I3I 

action  of  different  portions  of  tiie  solution  with  litmus,  alizarin,  and  Congo  red 
solution. 

4.  Boil  a  small  portion  of  the  nucleic  acid  with  about  10  c.c.  of  10  per  cent 
sulphuric  acid  for  one  to  two  minutes.     Divide  into  three  portions. 

(a)  To  one  portion  apply  carbohydrate  tests,  e.g.,  the  a-naphthol  fMolisch; 
reaction  and  Bial's  test.     What  do  these  indicate? 

(b)  To  a  second  portion  apply  a  test  for  purine  bases.  Add  an  excess  of 
ammonia  and  then  a  Uttle  silver  nitrate  solution. 

(c)  To  the  third  portion  apply  test  for  phosphate,  adding  ammonia  in  slight 
excess,  then  making  acid  with  nitric  acid,  adding  molybdic  solution  and  warming. 

9.  Preparation  of  Thymus  Nucleic  Acid.^ — "To  a  boiling  mi.xture  of  200  c.c.  of 
water,  10  grams  of  sodium  acetate  and  3.3  grams  of  NaOH,  is  added  in  small  suc- 
cessive portions  100  grams  of  trimmed  and  finely  ground  thymus  gland.  The  tissue 
usually  dissolves  completely  forming  a  pale  brown  liquid,  but  any  resistant  portions 
are  either  removed  or  gotten  into  solution  by  heating  for  a  short  time  over  a  small 
flame.  The  vessel  containing  the  products  is  now  immersed  in  a  briskly  boiling 
water-bath  where  it  is  allowed  to  remain  with  occasional  stirring  for  two  hours, 
when  the  product  is  diluted  with  one-third  its  volume  of  water  and  made  faintly 
but  distinctly  acid  to  litmus  with  50  per  cent  acetic  acid.  The  amount  of  acid 
required  is  about  10  c.c.  but  the  final  additions  must  be  made  with  care  because  the 
fluid  will  not  filter  unless  the  proper  condition  of  acidity  is  reached.  Any  difficulty 
met  at  this  point  may  be  easily  overcome  by  the  alternate  addition  of  acetic  acid  and 
sodium  hydroxide  and  testing  a  small  portion  of  the  material  after  each  addition  on 
a  small  flat  filter  that  has  been  heated  with  boiling  water.  When  the  acidity  has 
finally  been  obtained  which  is  favorable  to  rapid  filtration,  the  material  is  heated  to 
vigorous  boiling  and  filtered  with  a  hot  water  funnel.  Under  proper  conditions  the 
filtration  proceeds  with  considerable  rapidity  and  continuously  leaves  a  green  slime 
on  the  filter  and  gives  a  pale  yellow  filtrate  which  gelatinizes  upon  cooling.  The 
filtrate  and  washings  are  evaporated  on  a  water-bath  to  about  75  c.c  and  while  warm 
the  concentrated  solution  is  poured  slowly  into  100  c.c.  of  95  per  cent  alcohol.  On 
standing  over  night  the  precipitated  sodium  nucleate  settles  sharply  to  a  spongy 
white  mass  from  which  the  bulk  of  brown  alcoholic  fluid  can  be  sharply  decanted 
and  the  remainder  pressed  out  with  a  spatula  leaving  the  material  in  one  cohesive 
mass.  The  substance  is  washed  by  decantation  in  turn  with  80  per  cent  and  95  per 
cent  alcohol  and,  after  pressing  out  the  last  wash  fluid  as  far  as  possible  is  transferred 
to  a  flask  with  30  c.c  of  hot  water  and  heated  on  a  water-bath.  In  half  an  hour  or 
less,  insoluble  phosphates  will  collect  leaving  a  perfectly  transparent  interstitial 
fluid  which  is  treated  with  i  c.c.  of  20  per  cent  NaOH  to  lower  the  viscosity  and 
filtered  with  a  hot  water  funnel.  The  perfectly  transparent  yellow  filtrate  is  acidi- 
fied with  acetic  acid  and  poured  into  70  c.c.  of  95  per  cent  alcohol  when  sodium 
nucleate  wUl  be  precipitated  which  can  be  washed  by  decantation  as  before  with 
alcohol  of  increasing  strength  and  ground  in  a  mortar  with  absolute  alcohol  until 
it  has  crumbled  to  a  fine  white  powder.  If  necessary  the  absolute  alcohol  may  be 
decanted  and  renewed  once  or  twice  but  not  oftener  because  the  nucleate  emulsifies 
with  alcohol  after  the  last  traces  of  acetic  acid  and  sodium  acetate  have  been  washed 
away.  The  material  is  finally  .washed  on  a  filter  with  absolute  alcohol  and  allowed 
to  dry  in  a  sulphuric  acid  desiccator.  The  yield  of  nucleic  acid  is  about  ^.^  grams 
from  100  grams  of  gland.     The  product  is  a  fine  white  non-hygroscopic  powder 

^  From  Monograph  on  "Nucleic  Acids"  by  Waiter  Jones:  Longmans,  Green  &  Co. 


132  PHYSIOLOGICAL    CHEMISTRY 

that  can  scarcely  be  improved  by  any  method  of  purification.  It  is  a  soluble 
sodium  salt  of  thymus  nucleic  acid  but  is  generally  referred  to  simply  as  thymus 
nucleic  acid.  Very  similar  or  identical  substances  may  be  prepared  by  the  same 
procedure  from  other  animal  tissues,  rich  in  cell  nuclei  such  as  the  pancreas  and 
spleen." 

10.  Tests  on  Thymus  Nucleic  Acid. — 1-4.  Repeat  the  experiments  as  given 
under  yeast  nucleic  acid,  page  130.  5.  Make  a  4  per  cent  solution  of  thymus  nucleic 
acid  in  hot  water  {%  gram  to  10  c.c).  Allow  to  cool.  What  happens?  Divide 
into  two  portions.  To  one  add  a  little  NaOH  solution;  to  the  other  add  acetic  acid. 
Then  neutralize  carefully  in  each  case. 

Both  acetic  acid  and  NaOH  decrease  the  viscosity  of  the  nucleate  solution.  It 
may  be  changed  back  and  forth  from  the  gelatinous  to  the  fluid  condition  by  the 
alternate  addition  of  acid  and  alkali. 

11.  Tests  on  Purine  Bases  and  Derivatives. — (a)  Xanthine. — i.  Silver 
Nitrate  Reaction. — Dissolve  a  little  xanthine  in  ammonia  and  add  silver  nitrate 
solution.     Examine  a  little  of  the  precipitate  microscopically.     (See  page  351.) 

2.  Copper  Sulphate  Reaction. — Dissolve  a  little  of  the  substance  in  dilute 
alkali,  make  faintly  acid  with  acetic  acid.  Heat  to  boiling.  Add  i  c.c.  10 
per  cent  CuSO  4  and  then  a  few  drops  at  a  time  of  sodium  bisulphite  (saturated 
solution)  until  the  precipitate  becomes  yellowish.  All  of  the  purines  give  this 
reaction. 

3.  Nitric  Acid  Test. — Place  a  small  amount  of  the  substance  in  a  small 
evaporating  dish,  add  a  few  drops  of  concentrated  nitric  acid,  and  evaporate  to 
dryness  very  carefully  on  a  water-bath.  The  yellow  residue  upon  moistening 
with  caustic  potash  becomes  red  in  color  and  upon  further  heating  assvmies  a 
purplish-red  hue.  Now  add  a  few  drops  of  water  and  warm.  A  yellow  solution 
results  which  yields  a  red  residue  upon  evaporation.  Compare  with  similar 
reaction  on  other  purine  bases  and  uric  acid.     (See  Murexide  test,  Chapter  XXII. ) 

4.  Weidel's  Reaction. — Bring  a  small  amount  of  the  substance  into  solution  in 
bromine  water.  Evaporate  to  dryness  on  a  water-bath.  Remove  the  stopper 
from  an  ammonia  bottle  and  by  blowing  across  the  mouth  of  the  bottle  direct  the 
fumes  of  ammonia  so  that  they  come  into  contact  with  the  dry  residue.  Under 
these  conditions  the  presence  of  xanthine  is  shown  by  the  residue  assuming  a  red 
color.  A  somewhat  brighter  color  may  be  obtained  by  using  a  trace  of  nitric  acid 
with  the  bromine  water.  By  the  use  of  this  modification,  however,  we  may  get  a 
positive  reaction  with  bodies  other  than  xanthine. 

(b)  Hypoxanthine. — i.  Repeat  Experiments  i  and  3  under  Xanthine.  Ex- 
amine the  crystals  of  hypoxanthine  silver  nitrate  under  the  microscope.  (See 
page  351. J 

2.  Dissolve  a  little  of  the  substance  in  a  very  small  amount  of  hot  6  per  cent 
nitric  acid  and  allow  to  cool.  Characteristic  whetstone  crystals  of  hypoxanthine 
nitrate  should  be  formed.  Examine  under  the  microscope.  (See  Fig.  40,  page 
136.) 

(c)  Adenine. — i.  Warm  a  few  crystals  of  adenine  in  a  test-tube  with  a  little 
water.     They  should  become  cloudy  at  53°C. 

2.  Dissolve  a  httle  adenine  in  hot  water  and  add  a  few  drops  of  picric  acid. 
Examine  the  pale  yellow  crystals  under  the  microscope.  The  picrate  crystal- 
lizes as  needle  clusters. 

3.  Repeat  Experiment  3  under  Xanthine. 


NUCLEIC    ACIDS    AND    NUCLEOPROTEINS  1 33 

(d)  Guanine. — i.  Dissolve  a  little  substance  in  20  25  times  its  weight  of 
boiling  5  per  cent  alcohol.     Allow  to  cool  and  examine  crystals  microscopically. 

3.  Dissolve  a  little  guanine  in  20-25  times  its  weight  of  boiling  5  per  cent 
hydrochloric  acid.  Allow  to  cool  and  examine  crystals  under  microscope. 
(See  Fig.  39,  page  135.) 

3.  Perform  Experiment  3  under  Xanthine. 

(e)  Uric  Acid. — i.  On  a  small  amount  of  uric  acid  try  the  test  as  given  under 
Xanthine  number  3.     This  test  on  uric  acid  is  called  the  Murexide  test. 

2.  For  other  tests  on  uric  acid  see  Chapter  XXII  on  Urine. 

12.  Isolation  of  Guanine  and  Adenine  from  Nucleic  Acid  (Method  of  Wal- 
ter Jones). ^ — The  amino  purines  ma}-  be  isolated  from  yeasL  or  thymus  nucleic  acid 
or  from  glandular  tissue  (such  as  the  pancreas)  after  hydrolysis  of  the  material  with 
sulphuric  acid. 

In  the  case  of  yeast  nucleic  acid,  heat  10  grams  of  the  substance  with  50  c.c.  of 
10  per  cent  sulphuric  acid  on  a  boiling  water-bath  for  about  two  hours,  replacing 
any  water  lost,  or  using  a  condenser  tube.  To  the  hot  solution  add  concentrated 
ammonia  slowly  until  approximately  neutral.  Then  add  enough  excess  of  ammonia 
to  make  about  a  2  per  cent  solution.  Filter  off  the  precipitate  of  guanine  and  wash 
it  with  I  per  cent  ammonia.  Dissolve  in  as  small  an  amount  of  20  per  cent  sul- 
phuric acid  as  possible,  add  a  little  animal  charcoal  and  boil.  Filter,  heat  to  boiling 
and  precipitate  with  excess  of  ammonia.  Filter,  dry  the  precipitate  at  40°C.  and 
dissolve  it  in  about  20  parts  of  boiling  5  per  cent  hydrochloric  acid.  As  the  solu- 
tion cools  guanine  chloride  separates  out  as  needle-shaped  crystals.  Filter  off, 
wash  with  very  dilute  hydrochloric  acid  and  dry  in  the  air  (do  not  put  in  desiccator). 
Perform  the  nitric  acid  test  on  the  product. 

Combine  the  ammoniacal  filtrates  obtained  in  the  isolation  and  purification  of 
guanine.  Filter  if  necessary.  The  ammonia  may  then  be  boiled  off  and  an  excess 
of  picric  acid  added  in  which  case  a  yellow  precipitate  of  adenine  picrate  is  produced 
which  is  filtered  off  and  dried.  It  is  better,  however,  to  neutralize  the  ammonia  of 
the  combined  filtrates  and  make  faintly  acid  with  sulphuric  acid.  Then  precipitate 
the  adenine  as  its  copper  compound  (see  directions  under  experiment  on  Demonstra- 
tion of  Nucleases  B)  decomposing  this  with  hydrogen  sulphide  and  evaporating  the 
filtrate  from  the  copper  sulphide  to  dryness  on  the  water-bath.  Dissolve  the  residue 
in  hot  5  per  cent  sulphuric  acid  and  allow  to  crystallize  out.  If  necessary  dissolve 
in  hot  water  decolorize  with  a  little  charcoal  and  allow  to  crystallize  out  again. 
The  compound  has  the  formula  (C5H5N5)2.HnS04.2H20.  Apply  the  picric  acid 
and  nitric  acid  tests  as  given  under  adenine  (page  132). 

13.  The  Pyrimidine  Derivatives.  The  pyrimidine  derivatives, 
cytosine,  thymine,  and  uracil,  are  separated  from  nucleic  acid  witli 
some  difficulty.  'J1ie  following  test  may  be  made  on  a  solution  of 
cytosine  or  uracil.     Thymine  does  not  give  the  test. 

Wheeler-Johnson  Reaction  for  Uracil  and  Cytosine.  To  about  5  c.c.  of  the 
solution  under  examination  add  bromine  water  until  the  color  is  permanent. 
Avoid  the  addition  of  a  large  excess  as  this  will  interfere  with  the  test.  In 
case  the  solution  contains  only  small  quantities  of  cytosine  or  uracil  it  is  advis- 
able to  remove  any  excess  of  bromine  by  passing  a  stream  of  air  through  the 

1  See  Walter  Jones:  Alonograph  on  "  Xucleic  .\cids,"  1914,  Longmans,  Circen  &.  Co. 


134  PHYSIOLOGICAL   CHEMISTRY 

solution.     Now  add  an  excess  of  an  aqueous  solution  of  barium  hydroxide 
and  note  the  appearance  of  a  purple  color. 

Very  dilute  solutions  do  not  give  the  test.  Under  these  conditions 
the  solution  should  be  evaporated  to  dryness,  the  residue  dissolved  in  a 
little  bromine  water  and  the  excess  of  bromine  removed.  Then  upon 
adding  an  excess  of  barium  hydroxide  a  decided  bluish-pink  or  lavender 
color  will  appear  in  the  presence  of  as  small  an  amount  as  o.ooi  gram  of 
uracil. 

In  testing  solutions  for  cytosine  it  is  preferable  to  warm  or  boil  the 
solution  with  bromine  water,  and  after  cooling  the  solution  to  apply 
the  test  as  suggested  above,  being  careful  to  have  a  slight  excess  of 
bromine  present  before  adding  barium  hydroxide. 

14.  Demonstration  of  Nucleases  and  Purinases  in  Tissues.' — All 
glandular  tissues  contain  nucleic  acids  and  enzymes  capable  of  their 
hydrolysis  as  well  as  the  transformation  of  liberated  purine  bodies. 
By  allowing  autolysis  (self-digestion)  to  take  place  in  such  a  tissue  and 
studying  the  products  formed  it  is  possible  to  determine  what  enzymes 
were  present  in  the  tissue  under  examination.  Typical  results  may  be 
obtained  by  using  ox  and  pig  spleens,  which  differ  in  the  purine  enzymes 
which  they  contain.     The  two  experiments  should  be  run  in  parallel. 

A.  Preparation  of  the  Material  for  Digestion. — Run  the  gland  once  or  tmce 
through  a  meat  chopper.  Introduce  about  650  grams  into  a  two  liter  bottle,  fill 
about  three-quarters  full  of  water,  add  20  c.c.  of  chloroform,  and  allow  to  remain 
at  room  temperature  for  12-36  hours  with  occasional  agitation.  Strain  through 
linen  and  replace  the  turbid  extract  in  the  bottle  with  10  c.c.  of  chloroform.  This 
solution  contains  the  enzymes  and  nucleic  acid  of  the  tissue.  (Reserve  50  c.c. 
of  one  of  the  extracts  for  the  experiment  on  phosphonuclease  (E),  which  should 
also  be  started  at  this  time.)  The  bottle  is  tightly  closed  and  allowed  to  remain 
in  the  thermostat  4-5  days. 

B.  Separation  of  Purine  Derivatives  from  Other  Substances. — Introduce 
the  products  into  a  saucepan  or  large  evaporating  dish,  heat  to  brisk  boiling, 
make  faintly  alkaUne  with  caustic  soda,  boil  a  few  minutes,  make  faintly  acid  with 
acetic  acid,  boil  vigorously  and  filter  hot.  (If  desired  one-half  of  the  filtrate  may 
be  treated  with  100  c.c.  of  20  per  cent  sulphuric  acid  per  liter  and  boiled  for  one 
hour,  keeping  at  constant  volume.  At  the  end  of  the  hydrolysis  the  sulphuric  acid 
is  nearly  neutralized  with  caustic  soda  and  the  purine  bases  of  the  solution 
determined  as  in  the  other  half  of  the  filtrate.  This  will  give  information  as  to 
the  presence  of  deaminases  acting  upon  the  amino  purines  remaining  in  combina- 
tion). To  the  boiUng  filtrate  add  100  c.c.  of  10  per  cent  copper  sulphate  and  small 
successive  portions  (2-5  c.c.)  of  sodium  bisulphite  (commercial  saturated  solution) 
until  the  precipitate  takes  on  a  decided  yellow  color  due  to  precipitation  of  cuprous 
oxide.  Filter,  wash  the  precipitate  with  boiUng  water,  pierce  the  filter  and  wash 
the  precipitate  into  a  flask.    Add  i  per  cent  sodium  sulphide  solution  to  decom- 

1  From  Monograph  on  "Nucleic  Acids"  and  Laboratory  Notes  in  Physiological  Chem- 
stry  by  Professor  Walter  Jones  of  Johns  Hopkins  University,  with  additions. 


NUCLEIC   ACIDS   AND   NUCLEOPROTEINS 


135 


pose  the  copper  compound  continuing  the  addition  until  the  precipitate  becomes 
uniformly  black  and  then  in  small  successive  portions,  testing  a  drop  of  the  prod- 
uct after  each  addition  by  bringing  it  into  contact  with  a  drop  of  lead  acetate 
on  a  filter  paper.  To  the  boiUng  fluid  add  acetic  acid  (in  the  case  of  the  extract 
of  pig's  spleen  and  other  solutions  containing  guanine  the  acetic  acid  should  be 
replaced  by  dilute  sulphuric)  until  the  insoluble  copper  sulphide  collects  and  filter 
the  hot  fluid  as  quickly  as  possible. 

C.  Treatment  of  the  Filtrate  from  Pig's  Spleen. — When  the  filtrate  from  the 
copper  sulphide  is  cold  make  strongly  alkaUne  with  ammonia  and  precipitate  the 
purine  compounds  with  a  sUght  excess  of  ammoniacal  silver  nitrate.  Filter,  wash 
thoroughly  with  cold  water.  Pierce  the  paper,  wash  the  precipitate  into  a  flask 
with  boihng  water  and  decompose  the  silver  precipitate  with  hydrochloric  acid. 
When  enough  acid  has  been  added  the  silver  chloride  will  settle  as  a  heavy  case- 
ous precipitate  leaving  clear  interstitial  fluid.  Filter  and  heat  the  filtrate  to 
boiling.     Treat  with  an  excess  of  ammonia  (enough  to  make  about  1-2  per  cent). 


Fig.  39. — Guanine  Chloride. 
(Reproduced  from  crystals  furnished  by  Professor  Walter  Jones.) 

Allow  to  cool  and  filter  off  the  guanine  which  precipitates.  Wash  the  guanine 
with  I  per  cent  ammonia  and  then  suspend  it  in  a  Uttle  hot  water  and  add  a  few 
drops  of  20  per  cent  sulphuric  acid  to  dissolve  it.  At  the  boihng  point  add  a  Uttle 
animal  charcoal,  boil  and  filter.  Make  strongly  alkaline  with  ammonia.  Snow 
white  guanine  is  precipitated.     Dissolve  the  precipitate  in  20  volumes  of  boiling 

5  per  cent  hydrochloric  acid.  Upon  cooUng  beautiful  needle-shaped  crystals  of 
guanine  chloride  separate.     (See  Fig.  39.) 

Evaporate  the  filtrate  from  the  guanine  to  dryness  on  the  water-bath  to  expel 
ammonia.  Moisten  with  hydrochloric  acid  and  again  evaporate.  Treat  the 
residue  with  warm  water.  Does  it  dissolve  almost  completely  indicating  the 
absence  of  xanthine  and  uric  acid?  Test  a  drop  of  the  solution  with  picric  acid. 
If  no  precipitate  is  obtained  adenine  is  absent.  Evaporate  to  dryness,  moisten 
with  alcohol  and  again  evaporate.     Dissolve  the  residue  in  about  30  parts  of  hot 

6  per  cent  nitric  acid.  On  cooUng,  characteristic  whetstone  crystals  of  hypo- 
xanthine  nitrate  form.  ((See  Fig.  40,  page  136.)  The  xanthine  nitric  acid 
color  test  should  be  practically  negative  on  this  product. 


136 


PHYSIOLOGICAL    CHEMISTRY 


What  does  the  finding  of  guanine  and  hypoxanthine  but  not  adenine  or  xan- 
thine indicate  as  to  the  type  of  purine  deaminase  present  in  the  pig's  spleen? 

D.  Treatment  of  Filtrate  from  Ox  Spleen. — The  filtrate  from  the  copper  sul- 
phide should  be  evaporated  to  dryness  on  the  water-bath.  Extract  with  cold 
water.  Test  a  part  of  this  aqueous  solution  with  picric  acid.  A  lack  of  precipi- 
tate indicates  the  absence  of  adenine.  To  another  portion  add  anmionia  (a 
lack  of  precipitate  indicates  the  absence  of  guanine),  and  then  a  Uttle  ammo- 
niacal  silver  nitrate  solution  (lack  of  appreciable  precipitate  indicates  absence  of 
purines  of  any  kind  in  more  than  traces).  Dissolve  half  of  the  residue,  which 
should  consist  mainly  of  uric  acid  and  xanthine,  in  as  few  drops  of  concentrated 
sulphuric  acid  as  possible  and  dilute  with  4  volumes  of  water.  Stir  until  the 
uric  acid  begins  to  separate  and  then  let  stand  for  about  three  hours.  The  uric 
acid  is  completely  precipitated.  Apply  the  murexide  test.  To  the  remainder 
of  the  xanthine-uric  acid  residue  add  a  Uttle  4  per  cent  potassiimi  hydroxide  solu- 
tion.    Warm  and  add  an  equal  volume  of  30  per  cent  nitric  acid.     Allow  to  cool. 


Fig.  40. — Hypoxanthine  Chloride.^ 
(Reproduced  from  crystals  furnished  by  Professor  Walter  Jones.) 

Xanthine  nitrate  separates  out  in  a  granular  form,  showing  characteristic  crystals 

under  the  microscope.     Apply  the  nitric  acid  test. 

What  does  the  presence  of  uric  acid  and  xanthine  and  the  absence  of  guanine 
and  adenine  indicate  as  to  the  purine  enzymes  of  ox  spleen? 

E.  Demonstration  of  Nucleotidase  (Phosphonuclease). — In  this  experiment 
use  the  50  c.c.  portion  of  enzyme  solution  retained  from  Experiment  A  preceding. 
Prepare  a  2  per  cent  solution  of  yeast  nucleic  acid  aiding  the  solution  by  the  slow 
addition  of  KOH  solution  until  the  reaction,  as  indicated  by  a  few  drops  of  Utmus 
added,  is  neutral.  Prepare  a  series  of  three  large  test-tubes  as  follows :  In  No.  i 
place  10  c.c.  of  enzyme  solution  and  5  c.c.  of  nucleic  acid.  In  No.  2  place  10  c.c. 
of  enzyme  solution  and  5  c.c.  of  water.  In  No.  3  place  10  c.c.  of  enzyme  solution, 
boiled  and  5  c.c.  of  nucleic  acid.  To  each  tube  add  2-3  c.c.  each  of  CHCI3  and 
toluol  as  preservatives.  Place  the  tubes  at  37°C.  for  24  hours.  Add  i  c.c.  of 
litmus  solution  to  each  tube  and  note  whether  any  changes  in  reaction  have  taken 
place.  Put  the  tubes  in  boihng  water  for  a  few  minutes  to  coagulate  proteins 
Then  add  5  c.c.  of  5  per  cent  HCl  and  let  stand  for  an  hour.     This  precipitates. 

^Hypoxanthine  nitrate  crystallizes  in  similar  form. 


NUCLEIC   ACIDS   AND   NUCLEOPROTEINS  1 37 

any  unaltered  nucleic  acid  which  may  be  present.  Filter  and  take  an  aliquot 
portion  of  each  filtrate  (15  c.c).  Add  magnesia  mixture  and  a  few  cubic  centi- 
meters of  strong  ammonia.  Let  stand  over  night.  Any  phosphoric  acid  present 
will  be  precipitated  as  magnesium  ammonium  phosphate.  Observe  the  relative 
amounts  of  phosphate  in  each  case.  Has  any  phosphate  been  set  free  from  the 
nucleic  acid  added?     From  the  nucleic  acid  of  the  gland  extract? 

15.  Experiments  on  Uricase  (Uricolytic  Enzyme). — A.  Preparation  of  Extract 
Containing  Uricase. — Extract  about  50  grams  of  pulped  kidney  tissue  of  the  ox 
with  200  c.c.  of  toluol  or  chloroform  water  at  38°C.  for  24  hours,  with  occasional 
shaking.     Filter  and  use  the  filtrate  in  the  following  experiment. 

B.  Demonstration  of  Uricase. — Add  about  o.i  gram  of  uric  acid  to  10  c.c. 
of  water  and  bring  the  uric  acid  into  solution  by  the  addition  of  the  minimal  quan- 
tity of  KOH.  To  5  c.c.  of  this  uric  acid  solution  in  a  test-tube  add  50  c.c.  of  the 
uricoljrtic  enzjrme  extract  prepared  as  described  above.  Prepare  a  second  tube 
containing  a  like  amount  of  the  uric  acid  solution  but  boil  the  extract  before 
it  is  introduced.  Place  the  two  tubes  at  38°C.  for  two  days.  The  vessels  should 
be  open  to  the  air  and  the  contents  stirred  occasionally,  or  much  better,  a  con- 
tinuous current  of  air  which  has  gone  through  a  chloroform  wash  bottle  is  passed 
through  the  mixture.  Make  both  mixtures  faintly  acid  with  acetic  acid  and 
boil.  Filter  and  take  an  aUquot  of  each  filtrate.  Evaporate  to  low  volume,  make 
faintly  alkaUne  with  ammonia  and  filter.  Add  a  few  cubic  centimeters  of 
ammoniacal  silver  nitrate  solution.  Any  uric  acid  will  be  precipitated  as  silver 
urate.  The  control  should  give  a  heavy  precipitate  while  the  test  should  show 
no  precipitate  or  one  much  lighter  than  the  control,  due  to  uric  acid  destruction  in 
the  latter  case. 

If  it  is  desired  to  separate  out  the  pure  uric  acid  the  silver-purine  precipitate 
may  then  be  filtered  off.  It  is  washed  with  water  and  transferred  to  a  beaker  with 
the  aid  of  a  httle  water.  To  the  mixture  add  a  few  cubic  centimeters  of  hydrogen 
sulphide  solution  and  a  few  drops  of  HCl  and  allow  to  stand  over  night.  The  uric 
acid  should  separate  out  in  crystaUine  form  and  should  be  found  in  less  amount 
in  the  test  than  in  the  control  experiment.  The  uric  acid  may  also  be  titrated 
with  permanganate  as  in  the  FoUn-Shaflfer  method  for  uric  acid  in  urine.  See 
Chapter  XXVI  on  Quantitative  Analysis  of  Urine.)  This  will  enable  us  to 
determine  exactly  how  much  of  the  uric  acid  was  destroyed  through  the  action 
of  the  enzyme  extract. 


CHAPTER  VII 
GASTRIC  DIGESTION 

Gastric  digestion  takes  place  in  the  stomach  and  is  promoted  by 
the  gastric  juice,  which  is  secreted  by  the  glands  of  the  stomach  mucosa. 
These  glands  are  of  two  kinds,  fundus  glands  and  pyloric  glands  which 
are  situated,  as  their  names  imply,  in  the  regions  of  the  fundus  and 
pylorus.  The  principal  foods  acted  upon  in  gastric  digestion  are  the 
proteins  which  are  so  changed  by  its  processes  as  to  become  better  pre- 
pared for  further  digestion  in  the  intestine  and  for  their  final  absorption. 

From  reliable  experiments  made  upon  lower  animals  it  is  evident 
that  the  gastric  juice  is  secreted  as  the  result  of  stimuli  of  two  forms, 
i.e.,  psychical  stimuli  and  chemical  stimuli.  The  psychical  form  of 
stimuli  may  be  produced  by  the  sight,  thought,  or  taste  of  food,  and  the 
chemical  stimuli  may  be  produced  by  certain  substances,  such  as  water, 
milk,  the  extractives  of  meat,  etc.,  when  coming  in  contact  with  the 
stomach  mucosa.  The  stimulatory  power  of  water  has  been  very 
strikingly  demonstrated.^  The  claim  that  the  drinking  of  water  with 
meals  is  harmful  because  such  a  procedure  causes  a  dilution  of  the  gastric 
juice,  has  no  basis  in  fact.  The  drinking  of  water  with  meals  by  normal 
individuals  has  been  found  to  be  accompanied  by  a  more  economical 
utilization  of  the  ingested  proteins,  fats  and  carbohydrates.  Various 
other  desirable  and  no  undesirable  features  have  been  demonstrated 
as  accompanying  or  following  such  a  dietary  procedure.^  No  experi- 
mental evidence  has  been  submitted  which  can  justly  be  interpreted  as 
showing  any  harmful  influence  to  accompany  or  follow  the  drinking, 
by  normal  persons,  of  large  quantities  of  water  at  meal  time. 

The  volume  of  gastric  juice  secreted  during  any  given  period  of 
digestion  varies  with  the  quantity  and  kind  of  the  food.  These  con- 
clusions were  deduced  principally  from  a  series  of  so-called  delusive 
feeding  experiments.  A  dog  was  prepared  with  two  esophageal  open- 
ings and  a  gastric  fistula.     When  thus  prepared  and  fed  foods  of  various 

^  Foster  and  Lambert:  Journ.  Exper.  Med.,  lo,  820,  1908. 

Bergeim,  Rehfuss  and  Hawk:  Jour.  Biol.  Chem.,  19,  345,  1914. 
2  Hawk:  University  of  Pennsylvania  Medical  Bulletin,  18,  i,  1905. 

Fowler  and  Hawk:  Jour.  Exper.  Med.,  12,  388,  1910. 

Hattrem  and  Hawk:  Arch.  Int.  Med.,  7,  610,  1911. 

Mattill  and  Hawk:  Jour.  Am.  Chem.  Soc,  33,  pp.  1978,  1999,  and  2019,  1911. 

Hawk:  Arch.  Int.  Med.,  8,  382,  191 1. 

Hawk:  Proceedings  Soc.  Exp.  Biol,  and  Med.,  8,  36,  1910. 

Fairhall  and  Hawk:  Jour.  Am.  Chem.  Soc,  34,  546,  1912. 

Howe  and  Hawk:  Jour.  Biol.  Chem.,  11,  129,  1912. 

Hawk:  Biochem.  Bull.,  3,  420,  1914. 

138 


GASTRIC   DIGESTION  1 39 

kinds  such  as  meat  and  bread,  the  material  instead  of  passing  to  the 
stomach,  would  invariably  find  its  way  out  of  the  animal's  body  at  the 
upper  esophageal  opening.  Through  the  medium  of  the  gastric  fistula 
the  course  of  the  secretion  of  gastric  juice  could  be  carefully  followed. 
It  was  found  that  when  the  dog  ate  meat,  for  example,  there  was  a  large 
secretion  of  gastric  juice  notwithstanding  no  portion  of  the  food  eaten 
had  reached  the  stomach.  Further  experiments  made  through  the 
medium  of  a  cul-de-sac  formed  from  the  stomach  wall  have  given  us 
many  valuable  conclusions,  among  others  those  regarding  the  influence 
of  the  chemical  stimuli.  The  method  followed  was  to  feed  the  animal 
certain  substances  and  note  the  secretion  of  gastric  juice  in  the  miniature 
stomach  while  the  real  process  of  digestion  was  taking  place  in  the 
stomach  proper. 

Normal  gastric  juice  is  a  thin,  light  colored  fluid  which  is  acid  in 
reaction  and  has  a  specific  gravity  varying  between  i.ooi  and  i.oio. 
It  contains  only  2-3  per  cent  of  solid  matter  which  is  made  up  prin- 
cipally of  hydrochloric  acid,  sodium  chloride,  potassium  chloride,  earthy 
phosphates,  mucin  and  the  enzymes  pepsin,  gastric  rennin,  and  gastric 
lipase;  the  hydrochloric  acid  and  the  enzymes  are  of  the  greatest  impor- 
tance. The  acidity  of  the  gastric  juice  is  due  to /ree  hydrochloric  acid. 
It  was  formerly  believed  that  this  acid  was  secreted  by  the  parietal  cells 
of  the  fundus  as  well  as  by  the  chief  cells  of  both  the  fundus  and  pyloric 
glands.  It  has  been  claimed,^  however,  that  the  parietal  cell  is  the 
seat  oj  the  formation  of  the  hydrochloric  acid.  This  conclusion  is  based 
upon  the  formation  of  Prussian  blue  after  the  subcutaneous  injection  of 
potassium  ferrocyanide  and  ammonium  ferric  citrate  (rabbits  and 
guinea-pigs)  and  the  subsequent  (3  to  30  hours)  microscopical  examina- 
tion of  the  gastric  mucosa.  The  acid  was  shown  to  be  present  in  the 
lumina  of  the  gland  tubules  and  in  the  canaliculi  of  the  parietal  cells; 
traces  were  also  apparently  present  in  the  cytoplasm.  Later  Bensley 
and  Harvey-  showed  by  means  of  dyes  which  act  as  vital  stains  and 
as  indicators  very  sensitive  to  alkali  that  the  secretion  in  the  parietal 
cells  is  slightly  alkaline  whereas  that  in  the  lumen  of  the  gland  proper 
is  very  nearly  neutral.  Therefore,  the  acid  is  formed  entirely  above  the 
level  of  the  gland  proper,  i.e.,  in  the  foveolae  and  on  the  surface.  Ham- 
met^  and  still  more  recently  Macallum  and  Collip'*  have  confirmed  Miss 
Fitzgerald's  claim  that  the  acid  is  formed  in  the  parietal  cells. 

It  was  believed  that  hydrochloric  acid  was  generally  present  in  the 
gastric  juice  of  man  to  the  extent  of  about  0.2  per  cent.     Wlien  the 

^  Fitzgerald:  Proceedings  Royal  Society  (B),  83,  56,  1910. 

-  Bensley  and  Harvey:  Biological  Bulletin,  23,  225,  1912. 

'  Hammett:  Anatomical  Record,  g,  21,  1915. 

*  Reported  before  Society  of  Biological  Chemists,  Boston,  Dec.  27,  1915. 


I40  PHYSIOLOGICAL    CHEMISTRY 

amount  of  hydrochloric  acid  varied  to  any  considerable  degree  from  this 
value  a  condition  of  hypoacidity  or  hyperacidity  was  said  to  be  es- 
tablished. On  the  basis  of  more  recent  experiments/  however,  it 
appears  that  the  actual  acidity  of  the  gastric  juice  of  man  as  secreted 
by  the  glands  is  0.4  to  0.5  per  cent  hydrochloric  acid.  Boldyreff  be- 
lieves that  this  acidity  is  lowered  to  about  0.2  per  cent  by  regurgitation 
of  alkaline  fluid  from  the  intestine  (Chapter  VIII  on  Gastric  Analysis). 
Hydrochloric  acid  has  the  power  of  combining  with  protein  substances 
taken  in  the  food,  thus  forming  so-called  combined  hydrochloric  acid. 
This  combined  acid  is  a  less  potent  germicide  than  free  hydrochloric 
acid  and  has  less  power  to  destroy  the  amylolytic  enzyme  salivary 
amylase  (ptyalin)  of  the  saliva.  This  last  fact  explains  to  a  degree  the 
possibility  of  the  continuance  of  salivary  digestion  in  the  stomach. 

The  term  combined  hydrochloric  acid  is  really  a  misnomer.  WTien 
free  hydrochloric  acid  is  treated  with  a  protein  the  latter  functions  as 
a  base  and  a  salt  is  formed.  Therefore,  instead  of  having  ''com- 
bined hydrochloric  acid"  we  have  a  protein  salt  of  hydrochloric  acid. 
This  salt  ionizes  differently  from  the  free  acid.  This  fact  explains  the 
variation  in  the  germicidal  properties  of  the  two  solutions  as  well  as 
their  different  action  toward  enz\'mes,  such,  for  example,  as  salivary 
amylase  (see  page  61). 

The  hydrochloric  acid  of  the  gastric  juice  forms  a  medium  in  which 
the  pepsin  can  most  satisfactorily  digest  the  protein  food,  and  at  the 
same  time  it  acts  as  an  antiseptic  or  germicide  which  prevents  putre- 
factive processes  in  the  stomach.  It  also  possesses  the  power  of  inverting 
cane  sugar,  this  property  being  due  to  the  hydrogen  ion.  When  the 
hydrochloric  acid  of  the  gastric  juice  is  diminished  in  quantity  (hypoacid- 
ity) or  absent,  as  it  may  be  in  many  cases  of  functional  or  organic  dis- 
ease, there  is  no  check  to  the  growth  of  micro-organisms  in  the  stomach. 
There  are,  however,  certain  of  the  more  resistant  spores  which  even  the 
normal  acidity  of  the  gastric  juice  \\\\\  not  destroy.  A  condition  of 
hypoacidity  may  also  give  rise  to  fermentation  with  the  formation  of 
comparatively  large  amounts  of  such  substances  as  lactic  acid  and 
butyric  acid. 

The  question  of  the  origin  of  the  hydrochloric  acid  of  the  gastric 
juice  is  a  problem  to  whose  solution  many  investigators  have  given 
much  attention.  Many  theories  have  been  proposed,  among  them  the 
interaction  of  sodium  chloride  with  carbonic  acid,^  with  acid  phosphate^ 

^  Babkin:  Die  iiussere  Sekretion  der  Verdauungsdriisen,  Berlin,  1914,  p.  113. 

Boldyreff:  Quart.  Jour.  Exper.  Physiol.,  8,  i,  1914. 

Carlson:  Am.  Jour.  Physiol.,  38,  248,  19 15. 
^  Bunge:  Physiologic  and  Pathologic  Chemistry,  2nd.  Eng.  Ed.,  Philadelphia,  1902,  p.  135. 
^Maly:  Zeit.  f.  physiol.  Chem.,  i,  174,  1877.     ]Macallum  and  Collip:  Reported  before 
the  Society  of  Biological  Chemists,  Boston,  Dec.  27,  1915. 


GASTRIC   DIGESTION  141 

or  with  organic  acids.  On  the  other  hand,  it  is  possible  that  hydro- 
chloric acid  is  set  free  from*  combination  with  some  weak  base  as 
ammonia^  or  that  free  phosphoric  acid  valences  may  be  set  free  by  the 
enzymic  hydrolysis  of  organic  phosphoric  acid  esters.-  We  cannot  go 
into  a  discussion  of  these  various  theories.  That  the  hydrochloric 
acid  arises  from  the  chlorides  of  the  blood  appears  to  be  well  established 
but  the  same  cannot  be  said  with  regard  to  the  immediate  or  ultimate 
origin  of  the  hydrogen  ions  involved  in  the  reaction. 

The  most  important  of  the  enzymes  of  the  gastric  juice  is  the  pro- 
teolytic enzyme  pepsin.  The  pepsin  does  not  originate  as  such  in  the 
gastric  cells  but  is  formed  from  its  precursor,  the  z\  mogen  or  mother- 
substance  pepsinogen,  which  is  apparently  produced  by  the  parietal 
cells  of  the  fundus  as  well  as  by  the  chief  cells  of  the  fundus  and  pyloric 
glands.  Pepsinogen  may  be  differentiated  from  pepsin  from  the  fact 
that  it  is  more  resistant  to  alkali.'^  Upon  coming  into  contact  with  the 
hydrochloric  acid  of  the  secretion  this  pepsinogen  is  immediately  trans- 
formed into  pepsin.  Pepsin  is  not  active  in  alkaline  or  neutral  solutions, 
but  requires  at  least  a  faint  acidity  before  it  can  exert  its  power  to  dis- 
solve and  digest  proteins.  The  percentage  of  hydrochloric  acid  facili- 
tating the  most  rapid  peptic  action  varies  with  the  character  of  the 
protein  acted  upon,  e.g.,  0.08  per  cent  to  o.i  per  cent  for  the  digestion  of 
fibrin  and  0.25  per  cent  for  the  digestion  of  coagulated  egg-white. 
While  hydrochloric  acid  is  the  acid  usually  employed  to  promote  arti- 
ficial peptic  proteolysis,  other  acids,  organic  and  inorganic,  will  serve  the 
same  purpose.  Acidity  of  the  liquid  is  necessary  to  promote  the 
activity  of  the  pepsin,  but  the  acidity  need  not  necessarily  be  confined 
to  hydrochloric  acid. 

In  common  with  many  other  enzymes  pepsin  acts  best  at  about 
38°-4o°C.  and  its  digestive  power  decreases  as  the  temperature  is  low- 
ered, the  enzyme  being  only  slightly  active  at  o°C.  Its  power  is  only 
temporarily  inhibited  by  the  appHcation  of  such  low  temperatures, 
however,  and  the  enzyme  regains  its  full  proteolytic  power  upon  rais- 
ing the  temperature  to  40°C.  As  the  temperature  of  a  digestive  mix- 
ture is  raised  above  4o°C.  the  pepsin  gradually  loses  its  activity 
until  at  about  8o°-ioo°C.  its  proteolytic  power  is  permanently 
destroyed. 

Our  ideas  regarding  the  nature  of  the  products  formed  in  the  course 
of  peptic  proteolysis  have  undergone  considerable  revision  in  recent 
years.  The  former  view  that  these  products  included  only  acid  albu- 
minate  (acid  metaprotein),  proteoses,  peptones  and  peptides  is  no 

^Mathews:  Physiological  Chemistry,  New  York,  1915,  p.  374. 
-  Bergeim:  Proc.  Soc.  Exp.  Biol,  and  ^led.,  12,  21,  1914. 
^Langley:  Jour,  of  Physiol.,  3,  246. 


142  PHYSIOLOGICAL   CHEMISTRY 

longer  tenable.  From  the  investigations  of  numerous  observers  we 
have  learned  that  artificial  gastric  digestion  if  permitted  to  proceed  for  a 
sufficiently  long  period  will  yield,  in  addition  to  proteoses,  peptones  and 
peptides,  a  long  list  of  protein  cleavage  products  which  are  crystalline 
in  character,  including  leucine,  tyrosine,  alanine,  phenylalanine,  aspartic 
acid,  glutamic  acid,  proline,  leucinimide,  valine,  and  lysine.  A  similar 
group  of  substances  may  result  from  the  action  of  the  enzyme  trypsin 
(see  page  i86).  The  relative  amounts  of  proteoses,  peptones,  and  crys- 
talline substances  formed  depends  to  a  great  extent  upon  the  character 
of  the  protein  undergoing  digestion,  e.g.,  a  greater  proportion  of  pro- 
teoses results  from  the  digestion  of  fibrin  than  from  the  digestion  of 
coagulated  egg-white.  We  must  not  be  led  into  the  error  of  thinking 
that  the  large  number  of  protein  cleavage  products  just  mentioned  are 
formed  in  the  course  of  normal  gastric  digestion  within  the  animal 
organism.  They  are  formed  only  after  comparatively  long-continued 
hydrolysis.  In  pancreatic  digestion,  however,  there  are  formed  even 
under  normal  conditions  the  large  number  of  cleavage  products  to 
which  reference  has  been  made.  Peptic  proteolysis,  therefore,  within 
the  animal  organism  differs  from  tryptic  proteolysis  (see  page  i86) 
in  that  the  former  yields  larger  amounts  of  proteoses,  smaller  amounts 
of  peptones  and  no  considerable  quantity  of  crystalline  bodies  as  end- 
products  in  the  brief  period  during  which  proteins  are  ordinarily  sub- 
jected to  gastric  digestion.  Prolonged  hydrolysis  with  gastric  juice 
does,  however,  yield  considerable  quantities  of  the  non-protein  end- 
products.  In  cases  of  cancer  of  the  stomach  a  peptide-splitting 
enzyme  (erepsin)  is  said  to  be  present  in  the  stomach  contents.  This 
enzyme  is  believed  to  be  elaborated  by  the  cancer  tissue  and  its  identi- 
fication is  of  importance  in  connection  with  the  diagnosis  of  gastric 
cancer.  The  glycyl-trytophane  test^  is  sometimes  used  for  this 
purpose.  This  test  has  been  very  severely  criticized  (see  page  199). 
Abderhalden  and  Meyer^  have  shown  active  pepsin  to  be  present 
in  the  contents  of  all  parts  of  the  small  intestine.  It  is  suggested  that 
pepsin  may  be  adsorbed  in  the  stomach  by  such  protein  substances 
as  pass  into  the  intestine  in  solid  form  and  that  the  pepsin  thus  pro- 
tected may  bring  about  gastric  digestion  whenever  the  reaction  of  the 
surrounding  intestinal  contents  is  favorable.  This  fact  may  be  of 
importance  in  connection  with  the  profound  proteolysis  taking  place 
in  the  intestine.  Heretofore,  this  process  was  believed  to  be  furthered 
alone  by  trypsin  and  erepsin.  The  passage  of  adsorbed  pepsin  into  the 
intestine  may  be  an  efficient  aid  to  the  proper  digestion  of  solid  proteins 

^  Neubauer  and  Fischer:  Dent.  Arch.f.  klin.  Med.,  97,  499,  1909. 
'^Abderhalden  and  Meyer:  Zeit.  fur  physiol.  Chem.,  74,  67,  191 1. 


GASTRIC   DIGESTION  1 43 

which  are  ingested  without  sufficient  mastication  ("  bolted  ")  ^  and  which 
consequently,  at  times,  pass  into  the  intestine  in  rather  large  pieces 
(see  Chapter  XIV  on  Feces). 

Gastric  rennin,  the  second  enzyme  of  the  gastric  juice,  is  what  is 
known  as  a  milk  curdling  or  protein  coagulating  enzyme.  Rennin  acts 
upon  the  casein  of  the  milk,  splitting  it  into  a  peptone-like  body  and 
soluble  paracasein.  This  soluble  body,  in  the  presence  of  calcium 
salts,  combines  with  calcium,  forming  calcium  paracasein  which  is 
insoluble  and  precipitates.  There  is  some  uncertainty  regarding 
the  reaction  to  litmus  in  which  gastric  rennin  shows  the  greatest 
activity.  It  is,  however,  said  to  be  active  in  neutral,  alkaline,  or  acid 
solution.  However,  it  probably  possesses  its  greatest  activity  in  the 
presence  of  a  sUght  acid  reaction,  as  would  naturally  be  expected.  It 
is  especially  abundant  in  the  gastric  mucosa  of  the  calf,  and  is  used 
to  curdle  the  milk  used  in  cheese  making.  Gastric  rennin  is  always 
present  normally  in  the  gastric  juice,  but  in  certain  pathological  con- 
ditions such  as  atrophy  of  the  mucosa,  chronic  catarrh  of  the  stomach, 
or  in  carcinoma  it  may  be  absent. 

The  theory  that  the  proteolytic  activity  and  the  milk  curdling  prop- 
erty of  the  gastric  juice  reside  in  a  single  substance  is  causing  much 
controversy  at  the  present  time.  The  theory  was  originally  advanced 
by  the  Pawlow  school.^  According  to  Xencki  and  Sieber,^  the  milk 
curdling  and  protein  hydrolyzing  activities  reside  in  definite  and  distinct 
side  chains  of  a  single  mammoth  molecule.  The  view  which  has  rather 
the  strongest  support,  however,  is  to  the  eft'ect  that  there  are  two  entirely 
distinct  enzymes.  Important  evidence  has  been  advanced  in  favor  of 
this  view  by  Hammarsten,^  Taylor,^  and  Hemmeter.^  Burge^  has  re- 
ported experiments  upon  the  influence  of  a  direct  electric  current  upon 
solutions  possessing  typical  rennin  and  peptic  activities.  By  this 
means  he  was  able  to  prepare  a  solution  possessing  strong  rennin 
activity  but  entirely  devoid  of  peptic  activity.  This  furnishes  strong 
evidence  against  the  identity  of  the  two  enzymes  but  does  not  neces- 
sarily deny  the  accuracy  of  the  side-chain  theory. 

Gastric  lipase,  the  third  enzyme  of  the  gastric  juice,  is  a  fat-splitting 
enzyme.  It  possesses  but  slight  activity  when  the  gastric  juice  is  of 
normal  acidity,  but  evinces  its  action  principally  at  such  times  as  a 

^  Foster  and  Hawk:  Proceedings  of  the  Eighth  International  Congress  of  Applied  Chem- 
istry, New  York,  September,  191 2. 

^  Pawlow  and  Parastschuk:  Zeitschrift  fiir  Physiologische  Chemie,  42,  415,  1904. 
'  Nencki  and  Sieber:  Zeilschrift  fiir  Physiologische  Chemie,  23,  191,  1901. 
*Hammarsten:  Zeilschrift  fiir  Physiologische  Chemie,  56,  18,  1908;  94,  291,  1915. 

*  Taylor:  Journal  of  Biological  Chemistry,  5,  399,  1909. 

•  Hemmeter:  Berliner  klinischc  Wochenschrtft,  Ewald  Festnummer,  44,  1905. 
^  Burge:  American  Journal  of  Physiology,  29,  191 2. 


144  PHYSIOLOGICAL    CHEMISTRY 

gastric  juice  of  low  acidity  is  secreted  either  from  physiological  or  patho- 
logical cause.  The  digestion  of  fat  in  the  stomach  is,  however,  at 
most,  of  but  slight  importance  as  compared  with  the  digestion  of  fat  in 
the  intestine  through  the  action  of  the  lipase  of  the  pancreatic  juice 
(see  page  i88). 

Boldyreff^  has  shown  trypsin  to  be  present  in  stomach  contents,  due 
to  regurgitation  of  intestinal  contents  through  the  pylorus.  This  claim 
has  been  verified  by  others-  (see  Chapter  VIII  on  Gastric  Analysis) . 

PREPARATION  OF  AN  ARTIFICIAL  GASTRIC  JIHCE 

Dissect  the  mucous  membrane  of  a  pig's  stomach  from  the  muscular  portion 
and  discard  the  latter.  Divide  the  mucous  membrane  into  two  parts  (4/5  and 
1/5).  Cut  up  the  larger  portion,  place  it  in  a  large-sized  beaker  with  0.4  per  cent 
hydrochloric  acid  and  keep  at  38°-40°C.  for  at  least  24  hours.  Filter  ofif  the 
residue,  consisting  of  nuclein  and  other  substances,  and  use  the  filtrate  as 
an  artificial  gastric  juice.  This  filtrate  contains  pepsin,  rennin,  and  the  prod- 
ucts of  the  digestion  of  the  stomach  tissue,  i.e.,  acid  metaprotein  (acid  albu- 
minate), proteoses,  peptones,  etc. 

Preparation  of  a  Glycerol  Extract  of  Pig's  Stomach 

Take  the  one-fifth  portion  of  the  mucous  membrane  of  the  pig's  stomach  not 
used  in  the  preparation  of  the  artificial  gastric  juice,  cut  it  up  very  finely,  place  it 
in  a  small-sized  beaker  and  cover  the  membrane  with  glycerol.  Stir  frequently 
and  allow  to  stand  at  room  temperature  for  at  least  24  hours.  The  glycerol  will 
extract  the  pepsinogen.  Separate,  with  a  pipette  or  by  other  means,  the  glycerol 
from  the  pieces  of  mucous  membrane  and  use  the  glycerol  extract  as  required  in 
the  later  experiments. 

Products  of  Gastric  Digestion 

Into  the  artificial  gastric  juice,  prepared  as  above  described,  place  the  protein 
material  (fibrin,  coagulated  egg-white,  or  lean  beef)  provided  for  you  by  the 
instructor,  add  0.4  per  cent  hydrochloric  acid  as  suggested  by  the  instructor  and 
keep  the  digestion  mixture  at  40°C.  for  two  to  three  days.  Stir  frequently  and 
keep  free  hydrochloric  acid  present  in  the  solution  (for  tests  for  free  hydro- 
chloric acid  see  page  152). 

The  original  protein  has  been  digested  and  the  solution  now  contains  the 
products  of  peptic  proteolysis,  i.e.,  acid  metaprotein  (acid  albuminate),  proteoses, 
peptones,  etc.  The  insoluble  residue  may  include  nuclein  and  other  substances. 
Filter  the  digestion  mixture  and  after  testing  for  free  hydrochloric  acid  neutralize 
the  filtrate  with  potassium  hydroxide  solution.  If  any  of  the  acid  metaprotein 
(acid  albuminate)  is  still  untransformed  into  proteoses  it  will  precipitate  upon 
neutraUzation.  If  any  precipitate  forms  heat  the  mixture  to  boiling,  and  filter. 
If  no  precipitate  forms  proceed  without  filtering. 

1  Boldj'reff:  Quart.  Jour.  Exper,  Physiol.,  8,  i,  1914. 

^  Spencer,  Meyer,  Rehfuss  and  Hawk:  Am.  Jour.  Physiol,  39,  459,  1916. 


GASTRIC   DIGESTION  145 

We  now  have  a  solution  containing  a  mixture  consisting  principally  of  proteo- 
ses and  peptones.  Separate  and  identify  the  proteoses  and  peptones  according 
to  the  directions  given  on  pages  119  and  120. 

GENERAL  EXPERIMENTS  ON  GASTRIC  DIGESTION 

1.  Conditions  Essential  for  the  Action  of  Pepsin. — Prepare  four  test-tubes  as 
follows : 

(a)  Five  c.c.  of  pepsin  solution. 

(b)  Five  c.c.  of  0.4  per  cent  hydrochloric  acid. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

(d)  Two  or  3  c.c.  of  pepsin  solution  and  2-3  c.c.  of  0.5  per  cent  sodium  car- 
bonate solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  place  them  in  the  incu- 
bator or  water-bath  at  40°C.  for  one-half  hour,  carefully  noting  any  changes  which 
occur.  ^  (Carmine-fibrin  may  be  used  to  advantage  in  this  and  the  following  tests 
under  Gastric  Digestion.  In  this  case,  however,  the  experiments  should  be  con- 
ducted at  room  temperature.  For  directions  as  to  the  preparation  of  carmine- 
fibrin  see  Chapter  I.)  Now  combine  the  contents  of  tubes  (a)  and  fb)  and 
see  if  any  further  change  occurs  after  standing  at  40°C.  for  15-20  minutes.  Ex- 
plain the  results  obtained  from  these  five  experiments. 

2.  Influence  of  Different  Temperatures. — In  each  of  four  test-tubes  place  5 
c.c.  of  pepsin-hydrochloric  acid  solution.  Immerse  one  tube  in  cold  water  from 
the  faucet,  keep  a  second  tube  at  room  temperature  and  place  a  third  in  the 
incubator  or  water-bath  at  40°C.  Boil  the  contents  of  the  fourth  tube  for  a  few 
moments,  then  cool  and  also  keep  it  at  40°C.  Into  each  tube  introduce  a  small 
piece  of  fibrin  and  note  the  progress  of  digestion.  In  which  tube  does  the  most 
rapid  digestion  occur?     Explain  this. 

3.  The  Most  Favorable  Acidity. — Prepare  three  tubes  as  follows : 

(a)  Five  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution. 

(b)  Two  or  3  c.c.  of  0.2  per  cent  hydrochloric  acid  +  i  c.c.  of  concentrated 
hydrochloric  acid  +  5  c.c.  pepsin  solution. 

(c)  One  c.c.  of  0.2  per  cent  pepsin-hydrochloric  acid  solution  +  5  c.c.  of 
water. 

Introduce  a  small  piece  of  fibrin  into  each  tube,  keep  them  at  40'C.,  and  note 
the  progress  of  digestion.  In  which  degree  of  acidity  does  the  fibrin  digest  the 
most  rapidly? 

4.  Differentiation  between  Pepsin  and  Pepsinogen. — Prepare  five  tubes  as 
follows : 

(a)  Few  drops  of  glycerol  extract  of  pepsinogen  +  2-3  c.c.  of  water. 

(b)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  0.2  per  cent  hydro- 
chloric acid. 

(c)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  0.5  per  cent  sodium 
carbonate. 

^  Digestion  of  fibrin  in  a  pepsin-hydrochloric  acid  solution  is  indicated  first  by  a  swelling 
of  the  protein  due  to  the  action  of  the  acid,  and  later  by  a  disinlegration  and  dissolving  of 
the  fibrin  due  to  the  action  of  the  pepsin-hydrocliloric  acid.  If  uncertain  at  any  time 
whether  digestion  has  taken  place,  the  solution  under  examination  may  be  filtered  and  the 
biuret  test  applied  to  the  filtrate.  A  positive  reaction  will  signify  tlie  presence  of  acid 
metaprotein  (acid  albuminate),  proteoses  (albumoses),  or  peptones,  the  presence  of  any 
one  of  which  would  indicate  that  digestion  has  taken  place. 


146  PHYSIOLOGICAL    CHEMISTRY 

(d)  Two  or  3  c.c.  of  pepsin  solution  +  2-3  c.c.  of  i  per  cent  sodium  carbonate. 

(e)  Few  drops  of  glycerol  extract  of  pepsinogen  +  5  c.c.  of  i  per  cent  sodium 
carbonate. 

Add  a  small  piece  of  fibrin  to  the  contents  of  each  tube,  keep  the  five  tubes  at 
40°C.  for  one-half  hour  and  observe  any  changes  which  may  have  occurred.  To 
(a)  add  an  equal  volume  of  0.4  per  cent  hydrochloric  acid,  neutraUze  (c),  (d)  and 
(e)  with  hydrochloric  acid  and  add  an  equal  volume  of  0.4  per  cent  hydrochloric 
acid.  Place  these  tubes  at  4o°C.  again  and  note  any  further  changes  which  may 
occur.  What  contrast  do  we  find  in  the  results  from  the  last  three  tubes? 
On  the  basis  of  these  tests  what  is  the  relative  resistance  of  pepsin  and  pepsinogen 
to  alkaUs? 

5.  Comparative  Digestive  Power  of  Pepsin  with  Different  Acids. — Prepare  a 
series  of  tubes  each  containing  a  N/io  solution  of  one  of  the  following  acids: 
hydrochloric,  sulphuric,  nitric,  combined  hydrochloric,  acetic,  lactic  and  oxalic. 
To  each  acid  add  a  few  drops  of  the  glycerol  extract  of  pig's  stomach  and  a  small 
piece  of  fibrin.  Shake  well,  place  at  40°C.,  and  note  the  progress  of  digestion. 
In  which  tubes  does  the  most  rapid  digestion  occur? 

6.  Influence  of  MetaUic  Salts,  etc. — Prepare  a  series  of  tubes  and  into  each 
tube  introduce  4  c.c.  of  pepsin-hydrochloric  acid  solution  and  3-^  c.c.  of  one  of  the 
chemicals  listed  in  Experiment  18  under  Salivary  Digestion,  page  61.  Introduce  a 
small  piece  of  fibrin  into  each  of  the  tubes  and  keep  them  at  40°C.  for  one-half  hour. 
Note  the  variations  in  the  progress  of  digestion.  Where  has  the  least  rapid  diges- 
tion occurred? 

7.  Sahli's  Desmoid  Reaction. — This  is  a  method  for  testing  gastric  function 
without  using  the  stomach  tube.  The  underlying  principle  of  the  test  is  the  fact 
that  raw  cagtut  may  be  digested  in  gastric  juice  but  is  entirely  indigestible  in 
pancreatic  juice.  The  test  is  made  as  follows :  A  methylene -blue  pill  is  intro- 
duced into  a  small  rubber  bag  and  the  mouth  of  the  bag  subsequently  tied  with 
catgut.  1  The  small  bag  is  then  ingested  immediately  after  the  mid-day  meal  and 
the  urine  examined  5,  7,  9  and  18-20  hours  later  for  methylene  blue.  If  methyl- 
ene blue  is  present  in  appreciable  quantity,  it  will  impart  to  the  urine  a  greenish- 
blue  color.  If  not  present  in  sufficient  amount  to  impart  this  color  the  urine  should 
be  boiled  with  one-fifth  its  volume  of  glacial  acetic  acid,  whereupon  a  greenish- 
blue  color  results  if  the  chromogen  of  methylene  blue  is  present.  This  contin- 
gency seldom  arises,  however,  inasmuch  as  in  most  cases  of  uncolored  urine  it  will 
be  found  that  the  rubber  bag  has  passed  through  the  stomach  unopened.  If  the 
methylene  blue  is  found  in  the  urine  inside  of  18-20  hours  a  satisfactory  gastric 
function  is  indicated. 

For  Einhorn's  bead  method  for  the  study  of  digestive  function  see  chapter  on 
Feces. 

8.  Testing  the  Motor  and  Functional  Activities  of  the  Stomach. — This 
test  is  performed  the  same  as  Experiment  19  under  Salivary  Digestion,  page  61. 
If  the  experiment  was  carried  out  under  salivary  digestion  it  will  not  be  neces- 
sary to  repeat  it  here. 

'  About  0.05  gram  of  methylene  blue  is  mixed  with  sufficient  ext.  glycyrrhiza  to  form 
a  pill  about  3-4  mm.  in  diameter.  The  pill  is  then  placed  in  the  center  of  a  square 
piece  of  thin  rubber  dam  and  a  little  bag-like  receptacle  constructed  by  a  twisting 
movement.  The  neck  of  the  bag  is  then  closed  by  wrapping  three  turns  of  catgut  about 
it.  The  most  satisfactory  catgut  to  use  is  number  00  raw  catgut  which  has  previously 
been  soaked  in  water  until  soft.  When  ready  for  use  the  bag  should  sink  instantly  when 
placed  in  water  and  be  water-tight. 


GASTRIC   DIGESTION  1 47 

9.  Influence  of  Bile. — Prepare  three  tubes  as  follows : 

(a)  Five  cc.  of  pepsin-hydrochloric  acid  solution  -r  1/2-1  c.c.  of  bile. 

(b)  Five  c.c.  of  pepsin-hydrochloric  acid  solution  -f-  5  c.c.  of  bile. 

(c)  Five  c.c.  of  pepsin-hydrochloric  acid  solution. 

Introduce  into  each  tube  a  small  piece  of  fibrin.  Keep  the  tubes 
at  40°C.  and  note  the  progress  of  digestion.  Does  the  bile  exert  any 
appreciable  influence?-'     How? 

10.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of  five  tubes  as 
follows : 

(a)  Five  c.c.  of  fresh  milk  —  0.2  per  cent  hydrochloric  acid  add  slowly  until 
precipitate  forms  . 

(b)  Five  c.c.  of  fresh  milk  —  5  drops  of  rennin  solution.^ 

(c)  Five  c.c.  of  fresh  milk  —  10  drops  of  0.5  per  cent  sodium  carbonate  solu- 
tion. 

(d)  Five  c.c.  of  fresh  milk  —  10  drops  of  a  saturated  solution  of  ammonium 
oxalate. 

(e)  Five  c.c.  of  fresh  milk  —  5  drops  of  0.2  percent  hydrochloric  acid.  Now  to 
eachof  the  tubes  c  ,  d  ,and  'e;  adds  drops  of  rennin  solution.  Place  the  whole 
series  of  five  tubes  at  40^C.  and  after  10-15  minutes  note  what  is  occurring  in  the 
different  tubes.  Give  a  reason  for  each  partictilar  result.  How  do  ammonium 
oxalate  and  sodium  carbonate  prevent  coagulation? 

^  Any  good  commercial  rennin  or  rennet  preparation  may  be  used  in  preparing  this 
solution. 


CHAPTER  VIII 

GASTRIC  ANALYSIS 

The  method  of  gastric  analysis  which  has  been  in  vogue  clinically 
for  years  (see  page  174)  entails  the  feeding  of  a  standard  test  meal, 
the  removal  of  the  complete  stomach  contents  at  the  end  of  a  one- 
hour  period,  and  the  analysis  of  the  material  so  removed.  That  this 
method  is  inaccurate  has  been  repeatedly  demonstrated  in  the  author's 
laboratory^  and  elsewhere.^  Furthermore,  owing  to  the  bulk  of  the 
old  form  of  stomach  tube  and  the  discomfort  occasioned  by  its  use,  it 
is  impossible  to  follow  the  whole  cycle  of  digestion  and  estimate,  step 


Time 


Ihr. 


2hr. 


Fig.  41. — Normal  and  Pathological  Curves  after  an  Ewald  Meal. 

I.  normal  curve;  2  delayed  digestion  with  late  hyperacidity;  3,  larval  hyperacidity; 
4,  tardive  hyperacidity;  5,  marked  continued  secretion  from  obstruction. 

by  step,  the  exact  changes  which  take  place  in  the  stomach  after  the 
introduction  of  definite  food  mixtures  into  that  organ. 

Realizing  the  inadequacy  of  the  procedure  entailed  in  the  old 
method  of  gastric  analysis,  a  new  procedure  has  been  developed  by  Dr. 
Martin  E.  Rehfuss  in  the  author's  laboratory.  This  so-called  "Frac- 
tional Method"  entails  the  analysis  of  samples  of  material  withdrawn 
from  the  stomach  (by  syringe)  at  short  intervals  for  a  period  of  two 
hours  or  more  (until  stomach  is  empty)  after  the  ingestion  of  the  test 
meal.     By  this  means  the  observer  is  able  to  follow  the  entire  cycle  of 

'  Rehfuss:  Jour.  Am.  Med.  Ass'n,  64,  569,  1914. 

Rehfuss,  Bergeim  and  Hawk:  Jour.  Am.  Med.  Ass^n,  63,  909,  1914. 

Bergeim,  Rehfuss  and  Hawk:  Jour.  Am.  Med.  Ass^n,  63,  11,  1914. 
*  Harmer  and  Dodd:  Arch.  Int.  Med.,  Nov.  13,  1913,  p.  488. 

148 


GASTRIC   ANALYSIS 


149 


gastric  digestion  and  is  not  limited,  as  in  the  old  method,  to  information 
derived  from  the  analysis  of  a  single  sample  of  stomach  contents  with- 
drawn at  the  end  of  one  hour.  That  the  acid  values  obtained  by  the 
old  method  may  be  grossly  misinterpreted  and  lead  to  an  incorrect 
diagnosis  is  indicated  by  the  foregoing  diagram  (Fig.  41): 

It  is  set  forth  in  the  above  diagram  that  various  t>'pes  of  abnormal 
gastric  secretion  would  be  considered  normal  on  the  basis  of  a  single 
examination  at  the  end  of  one  hour  whereas  the  application  of  the 
fractional  method  reveals  the  abnormality  of  the  secretion  and  enables 
a  rapid  and  correct  diagnosis.  The  removal  of  samples  of  gastric 
contents  at  short  intervals,  for  a  period  of  two  hours  or  more  after  a 
test  meal,  is  made  possible  by  the  use  of  a  modified  stomach  tube^  of 
small  diameter  (No.  12  French  tubing)  and  fitted  with  a  metal  tip. 


Fig.  42. — Rehtuss  Stomach  Tube. 


The  tip  is  slotted  ^^'ith  large  perforations,  the  diameter  of  each  being 
equivalent  to  the  maximum  bore  of  the  tubing.  Such  a  tube  can  be 
left  in  the  stomach  through  the  entire  cycle  of  gastric  digestion  \Nithout 
inconvenience  to  the  patient.-  A  cut  of  the  Rehfuss  stomach  tube 
(Fig.  42)  is  shown  above. ^ 

The  idea  of  making  a  fractional  examination  of  gastric  contents  is 
not  new.  Most  of  such  attempts  have  been  made,  however,  by  using 
the  old  t^-pe  of  stomach  tube  and  removing  the  entire  stomach  contents 
at  different  intervals  on  successive  days,  e.g.,  after  fifteen  minutes  the 
first  day,  thirty  minutes  the  second  day,  forty-five  minutes  the  third 
day,  etc.     Hayem*  was  the  first  to  employ  this  method  and  later 

1  Rehfuss:  Am.  Jour.  Med.  ScL,  June,  1914. 

^  McClendon  has  recently  suggested  the  introduction  of  an  electrode  into  the  stomach  in 
an  attempt  to  follow  the  consecutive  changes  in  the  hydrogen  ion  concentration  of  the 
stomach  contents  (see  .4w.  Jour.  Physiol.,  38.  180,  1915). 

'  This  tube  is  manufactured  by  Charles  Lentz  &  Sons,  Philadelphia. 

*  Hayem:  Brouardel  &  Gilbert's,  Traitc  dc  Mcdcciue,  4,  236,  1905. 


I50 


PHYSIOLOGICAL    CHEMISTRY 


Ewald  and  Boas/  Reichmann,-  v.  Jaksch,^  Kornemann,^  Schule^  and 
Gregersen^  followed  a  similar  procedure.  The  first  report  of  data  from 
the  entire  gastric  cycle  obtained  by  means  of  a  small  bore  tube  were 
made  by  Ehrenreich.'^  This  investigator  used  a  Nelaton  catheter. 
Skaller^  has  reported  a  few  experiments  in  which  a  small  bore  stomach 
tube  with  a  metal  tip  was  employed  and  the  stomach  contents 
subjected  to  fractional  analysis. 

Until  recent  years,  the  concensus  of  opinion  based  principally  upon 
the  work  of  the  Pawlow  schooP  was  to  the  effect  that  the  gastric  juice 


Total  additij 


Frceacufity 


Trgpsln 


I20niinates 


Diet.  100cx:.o^ 0.542 7oHCl  at23X 

F  G,  43. — Influence  of  Acid  Introduced  into  the  Normal  Human  Stomach. 
{Spencer,  Meyer,  Rehfiiss  and  Hawk:  American  Journal  of  Physiology,  March,  1916.) 

of  normal  man  had  an  average  acid  concentration  of  0.2  per  cent 
hydrochloric  acid,  whereas  the  gastric  juice  of  the  dog  and  cat  had  an 
average  acid  concentration  of  0.4-0.5  per  cent  hydrochloric  acid. 
These  experiments  were  based  principally  upon  the  examination  of 
the  pure  gastric  juice  of  the  lower  animals  as  compared  with  the  stomach 

'  Ewald  &  Boas:   Virchow's  Arch.,  loi,  325,  1885. 
2  Reichmann:  Zeit.  f.  klin.  Med.,  24,  565,  1885. 
*  V.  Jaksch:  Zeit.  f.  klin.  Med.,  19,  383,  1890. 
*Kornemann:  Arch.  f.  Verdauungskr.,  p.  369,  1912. 
'Schule:  Zeit.  f.  klin.  Med.,  27,  461,  1895. 
*Gregersen:  Arch.  f.  Verdauungskr.,  19,  263,  1913. 
^  Ehrenreich:  Zeit.f.  klin.  Med.,  p.  231,  1912. 
^Skaller:  Berl.  klin.  Woch.,  50,  No.  47,  1913. 

»  Pawlow:  The  Work  of  the  Digestive  Glands.     Translated  by  Thompson,  Second  Edition, 
1910. 


GASTRIC    ANALYSIS 


151 


contents  of  man.  Later  experiments'  have,  however,  demonstrated 
that  the  acid  concentration  of  the  freshly  secreted  gastric  juice  of  man 
is  similar  to  that  of  the  dog,  i.e.,  0.4-0.5  per  cent.  Boldyreff  claims 
that  this  initial  high  acidity  of  the  human  gastric  juice  is  normally 
lowered  to  the  "optimum  acidity"  of  0.15-0.2  per  cent  hydrochloric 
acid  by  regurgitation  of  alkaline  fluids  (bile,  pancreatic  and  intestinal 
juices)  from  the  intestine.  This  claim  has  been  substantiated  by 
experiments  made  in  the  author's  laboratory-  and  elsewhere.'^  Both 
bile  and  tr>'psin  are  easily  identified  in  the  stomach  contents  of  man 
after  the  introduction  of  0.5  per  cent  hydrochloric  acid  into  the  empty 
organ.  The  above  points  are  illustrated  by  the  chart  shown  in  Fig. 
43,  page  150." 

The  composition  of  human  gastric  juice  and  of  the  residuum  (see 
page  160)  is  given  in  the  following  table: 


COMPOSITION  OF  HUMAN  GASTRIC  JUICE. 


Constituent 

Appetite  juice^ 

Residuum' 

Specific  gravity 

1 .007 

1.006 

A  degrees  

-0-55 

-0.47 

Total  acidity,  per  cent 

0.45                                           0.30 

Per  100  CO.  of  Juice                                                           j 

1 

Total  solids,  gram 

Organic  solids,  gram 

0.55                         j                 0.98^ 

0.41 

0.53^ 

Inorganic  solids,  gram 

0.14 

0.45^ 

Total  nitrogen,  gram 

0.060 

0.066^ 

Total  phosphorus,  gram 

0.005^ 

Total  sulphur,  gram 

0.007^ 

Ammonia  N,  gram 

0.002-3 

Amino-acid  N,  gram 

0 . 003-9 

Chlorides,  gram 

0-5 

1 

1  Babkin:  Die  Aussere  Sekretion  der  Verdauungsdriisen,  Berlin,  1914. 

Boldyreff:  Transactions  of  nth  Congress  of  Physicians,  St.  Petersburg,  1909. 

Boldyreff:  Quart.  Jour.  Exp.  Med.,  8,  i,  19 14. 

Carlson:  Am.  Jour.  Physiol.,  38,  248,  1915. 

Bergeim,  Rehfuss  &  Hawk:  Jour.  Biol.  Client.,  19,  345,  19 14. 
-  Spencer,  Meyer,  Rehfuss  and  Hawk:  Ai>i.  Jour.  Physiol.,    39,  459,  1916. 
*Migai:  Diss.,  St.  Petersburg,  1909. 

Milosorov:  Zent.  Physiol.,  28,  615,  1914. 

Zaitzeff:  Russky  Vrach.,  14,  No.  29,  191 5. 

*  Spencer  et  al:  Loc.  cit. 

*  Carlson:  Loc.  cit. 

*  Fowler,  Rehfuss  and  Hawk:  Loc.  cit. 
'Fowler  &  Buchanan:   Unpublished. 


152  physiological  chemistry 

The  Use  of  Indicators  in  Determining  the  Reaction  of  Gastric 
Juice  and  Other  Fluids 

The  reaction  of  the  gastric  juice  and  other  body  fluids  is  most 
readily  tested  by  means  of  indicators,  so-called  because  they  show 
changes  of  color  with  differing  degrees  of  acidity  or  alkalinity  of  the 
solution.  They  behave  as  though  they  were  weak  acids  or  bases  whose 
ions  and  unionized  molecules  have  different  colors.  Modern  theories 
of  color  in  organic  compounds  however  class  them  as  tautomeric 
substances. 

A  neutral  solution  is  one  in  which  there  are  equal  numbers  of  hy- 
drogen and  hydroxyl  ions.  An  acid  solution  has  a  preponderance  of 
hydrogen  ion  and  an  alkaline  solution  an  excess  of  hydroxyl  ion.  All 
indicators  do  not  show  changes  of  color  at  the  true  neutral  point,  but 
at  some  fixed  degree  of  acidity  (or  alkalinity),  i.e.,  at  a  definite  hydrogen 
or  hydroxyl  ion  concentration.  Indicators  which  change  color  at  the 
approximate  true  neutral  point  are  litmus  and  rosolic  acid,  while  phenol- 
phthalein  changes  color  in  a  slightly  alkaline  solution.  Congo  red, 
sodium  alizarin  sulphonate  and  tropaeolin  00  are  examples  of  indicators 
which  change  color  in  an  acid  solution. 

Organic  acids  in  general  are  not  sufficiently  strong;  i.e.,  do  not  dis- 
sociate with  the  production  of  enough  hydrogen  ion  to  cause  color 
changes  in  dilute  solution  with  indicators  of  the  last-mentioned  class. 
Litmus,  rosolic  acid  and  phenolphthalein,  however,  change  at  so 
low  a  hydrogen  ion  concentration  that  they  are  affected  by  dilute 
solutions  of  organic  acids  and  may  be  used  for  their  titration.  Even 
very  dilute  solutions  of  mineral  acids  are  sufficiently  acid  to  produce 
color  changes  with  Congo  red,  alizarin,  etc.,  and  hence  these  indicators 
may  be  used  in  the  titration  of  mineral  acid.  Phenolphthalein  which 
changes  color  in  a  weakly  alkaline  solution  indicates  the  presence  of  acid 
combined  with  weakly  alkaline  substances  (as  protein)  as  well  as  other 
types  of  acid  such  as  acid  salts,  and  hence,  is  used  in  the  titration  of 
solutions  for  their  total  acidity. 

The  hydrogen  ion  concentration  of  pure  water  or  a  neutral  solution 
is  approximately  iXio~^,  being  expressed  as  approximate  moles  of 
hydrogen  ion  per  liter.  That  is  water  is  a  1/10,000,000  N  solution  of 
hydrogen  ions.  The  concentration  of  hydroxyl  ions  in  pure  water  or  a 
neutral  solution  is  exactly  equal  to  that  of  the  hydrogen  ions,  so  that 
water  may  be  considered  to  be  an  N/ 10,000,000  alkali  as  well  as  an 
N/io,ooo,ooo  acid.  Hydrogen  ion  concentrations  are  often  ex- 
pressed for  the  sake  of  brevity  as  their  logarithms  with  the  sign  re- 
versed.    For   example   the   logarithm   of  iXio~^  would  be  -7.0  and 


GASTRIC   ANALYSIS  I  53 

according  to  this  notation  the  H  ion  concentration  would  be  expressed 
as  Ph  =  7-o.  The  product  of  the  hydrogen  ion  concentration  (H"^) 
by  the  hydroxyl  ion  concentration  (OH")  is  constant  at  about  iXio~^* 
so  that  as  (H"^)  increases  from  iXio"^  (Ph  =  7-o)  to  iXio"'* 
(Ph  =  4.0)  the  (0H~)  falls  to  iXio~^°,  and  vice  versa.  It  must  be 
borne  in  mind  that  higher  figures  for  the  logarithmic  notation  indicate 
lower  figures  for  (H"^).  The  hydrogen  ion  concentrations  at  which 
certain  indicators  commonly  used  in  titration  work  change  color,  are 
indicated  below. 

True  nature 
Indicator  Hydrogen  ion  concentration  .,       ., 

color  changes 

Phenolphthalein Between  iXio~*  and  iXio"^ Alkaline. 

Neutral  red i  X  io~^ Xeutral. 

Rosolic  acid i  X  lo"' Xeutral. 

Litmus Between  iXio"^  and  iXio"^ Xeutral. 

Sodium  alizarin  sulphonate Between  iXio~*  and  iXio~^ Acid. 

Congo  red Between  i  X  lo"^  and  i  X  io~^ .\cid. 

Dimethyl-amino-azobenzene Between  iXio"^  and  iXio"'' Acid. 

Methyl  orange Between  iXio"^  and  iXio~^ Acid. 

Tropajolin    00 iXio~- Acid. 

Tests  with  Indicators. — Prepare  a  series  of  solutions  of  varying  acidities  as 
outlined  in  the  following  table,  page  154.  Introduce  5  or  10  c.c.  portions  of  each 
of  these  into  a  series  of  test-tubes  and  add  to  each  a  few  drops  of  a  solution  of 
Tropaeolin  00.  Make  a  note  of  the  colors  produced,  in  the  spaces  left  for  this 
purpose.  In  the  same  way  test  out  the  other  indicators  mentioned,  in  order, 
using  in  each  case  a  few  drops  of  the  indicator  solution.  The  tests  using  the  last 
three  mentioned  indicators:  Giinzberg's,  Boas'  and  TropaeoUn  (evaporation 
test)  are  carried  out  differently  as  indicated  on  page  155. 

Are  the  following  assumptions  on  which  the  use  of  certain  of  these 
indicators  in  gastric  analysis  is  based  borne  out  by  your  findings : 

1.  That  Topfer's  reagent  (Dimethyl-amino-azo-benzene)  gives  its 
characteristic  pinkish-red  color  only  in  the  presence  of  free  HCl. 

2.  That  a  blue  color  with  Congo  red  indicates  free  hydrochloric  (or 
other  mineral  acid),  a  violet  color  indicates  an  organic  acid,  and  a  brown 
color  indicates  combined  hydrochloric  acid. 

3.  That  Tropaeolin  00  and  methyl  orange  are  indicators  for  free 
mineral  acid. 

4.  That  alizarin  reacts  to  free  mineral  acid,  organic  acids  and  acid 
salts  but  not  to  combined  HCl. 

5.  That  phenolphthalein  can  be  used  in  titrating  total  acidity,  that 
is,  acidity  due  to  mineral  and  organic  acids,  acid  salts  and  combined 
acid. 

6.  That  iodine  is  liberated  from  KI-KIO3  to  a  relatively  slight  ex- 
tent by  other  than  free  mineral  acid. 


154 


PHYSIOLOGICAL    CHEMISTRY 


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GASTRIC   ANALYSIS  I  55 

7.  That  Giinzberg's  test  is  the  most  satisfactory  one  for  free  HCl 
and  that  Boas'  reagent  and  Tropa^olin  00  are  also  delicate  reagents  for 
free  mineral  acid. 

Special  Tests  for  Free  HCl.  Perform  the  following  tests  on  the  solutions  as 
outhned  on  page  154  and  tabulate  the  results. 

1.  Giinzberg's  Reagent.' — Place  1-2  drops  of  the  reagent  in  a  small  porcelain 
evaporating  dish  and  carefully  evaporate  to  dryness  over  a  low  flame.  Insert 
a  glass  stirring  rod  into  the  mixture  to  be  tested  and  draw  the  moist  end  of  the 
rod  through  the  dried  reagent.  Warm  again  gently  and  note  the  production  of  a 
purphsh-red  color  in  the  presence  of  free  hydrochloric  acid. 

2.  Boas'  Reagent.- — Perform  this  test  in  the  same  manner  as  i,  above. 
Free  hydrochloric  acid  is  indicated  by  the  production  of  a  rose-red  color  which 
becomes  less  pronounced  on  cooling. 

3.  Tropaeolin  OO,' 

NH  (CeHs)  _C6H4-N  =  N-C6H4-S03Na. 

Place  2  drops  of  the  solution  to  be  tested  and  i  drop  of  the  indicator  in  an  evapo- 
rating dish  and  evaporate  to  dryness  over  a  low  flame.  The  formation  of  a  red- 
dish-violet color  indicates  free  hydrochloric  acid. 

This  test  may  also  be  conducted  in  the  same  manner  as  i,  above. 

Hydrogen  Ion  Concentration  and  Titratable  Acidity 

The  acidity  of  a  solution  may  be  determined  in  two  different  ways 
by  means  of  indicators.  One  method  is  by  titration  with  standard 
alkali  using  the  indicator  to  determine  the  end  point  of  the  titration. 
For  this  purpose  the  indicator  should  be  one  which  gives  a  sharp  color 
change  which  is  sensitive  to  the  form  of  acidity  which  is  to  be  deter- 
mined, and  which  is  not  destroyed  by  any  substance  contained  in  the 
titration  mixture.  Thus  phenolphthalein  can  be  used  for  the  titration 
of  strong  bases  and  nearly  all  weak  acids,  but  cannot  be  used  for  weak 
bases,  and  is  unsatisfactory  in  the  presence  of  ammonium  salts.  Methyl 
orange  on  the  other  hand  is  useful  for  strong  acids  and  weak  bases  such 
as  ammonia  and  for  the  soluble  carbonates  but  cannot  be  used  for  weak 
acids  such  as  carbonic  acid  or  the  organic  acids.  Almost  any  indicator 
may  be  used  in  the  titration  of  mineral  acids  against  strong  bases  such 
as  KOH  inasmuch  as  under  these  conditions  i  drop  of  the  standard 
solution  will  throw  the  hydrogen  ion  concentration  so  far  beyond  that  of 
neutrality  that  the  turning  point  of  any  common  indicator  ^v'ill  be 
passed. 

Titration  does  not,  however,  enable  us  to  determine  in  all  cases  the 

*  Giinzberg's  reagent  is  prepared  by  dissolving  2  grams  of  phloroglucinol  and  i  gram  of 
vanillin  in  loo  c.c.  of  95  per  cent  alcohol. 

^  Boas'  reagent  is  prepared  by  dissolving  5  grams  of  resorcinol  and  3  grams  of  sucrose  in 
100  c.c.  of  50  per  cent  alcohol. 

^  Prepared  by  dissolving  0.05  gram  of  tropsolin  00  in  100  c.c.  of  50  per  cent  alcohol. 


156  PHYSIOLOGICAL   CHEMISTRY 

true  acidity  of  a  solution,  that  is,  its  hydrogen  ion  concentration.  In  the 
case  of  strong  acids  and  bases  very  accurate  results  for  the  true  acidity 
may  be  obtained  in  this  way.  In  the  case  of  weak  acids  or  bases  the 
titration  values  may  give  but  slight  information  as  to  the  true  acidity. 
Thus  in  the  case  of  a  slightly  dissociated  acid,  such  as  acetic  acid,  as 
fast  as  the  acidity  due  to  its  dissociated  hydrogen  ions  is  neutralized 
the  undissociated  acid  ionizes  further  and  the  titration  value  finally 
obtained  represents  the  total  acid  present  at  the  beginning  both  ionized 
and  unionized.  Salts  of  strong  acids  and  very  weak  bases  and  vice 
versa  also  hydrolyze  during  the  course  of  the  titration  and  the  values 
obtained  in  no  sense  represent  the  true  acidity. 

Hydrogen  ion  concentrations  may  be  determined  through  a  certain 
range  by  means  of  indicators.  The  unknown  solution  is  treated  with 
a  few  drops  of  indicator  and  the  color  obtained  compared  with  that 
produced  with  the  same  amount  of  indicator  and  a  solution  of  known 
hydrogen  ion  concentration.  If  the  same  tint  is  produced  in  both 
cases  the  two  acidities  are  the  same.  This  is  of  course  only  true  when 
the  indicator  chosen  is  a  suitable  one,  that  is,  one  that  shows  definite 
color  changes  in  hydrogen  ion  concentrations  in  the  neighborhood  of 
that  of  the  unknown.  The  choice  of  indicators  for  this  purpose  is 
somewhat  different  than  that  for  titration  purposes.  For  use  in  the 
determination  of  the  hydrogen  ion  concentration  of  a  solution  we  need 
an  indicator  showing  a  very  gradual  change  in  color  through  a  given 
range,  one  which  is  not  readily  affected  by  the  presence  of  neutral  salts 
or  other  substances  likely  to  be  present,  and  the  color  of  which  does 
not  fade  too  rapidly.  The  ranges  through  which  a  number  of  indicators 
may  be  used  with  satisfactory  results  for  the  determination  of  hydrogen 
ion  concentrations  is  indicated  in  the  chart  (Fig.  44,  page  157).  Those 
surrounded  by  the  heavy  lines  are  the  most  satisfactory. 

The  chart  also  indicates  how  standard  solutions  of  definite  hydrogen 
ion  concentrations  may  be  made  up  from  a  series  of  stocK.  solutions, 
by  mixing  in  various  proportions.  The  stock  solutions  indicated  on  the 
chart  were  suggested  by  Sorensen  and  are  as  follows:  o.ioNHCl; 
o.ioNNaOH;  7.505  g.  glycocoll  plus  5.85  gm.  NaCl  per  liter;  11.876 
g.  Na2HP04.2H20  per  liter;  9.078  g.  KH2PO4  per  liter;  21.008  g. 
citric  acid  in  i  liter  of  0.20  N  NaOH;  12.404  g.  boric  acid  in  i  liter  of 
o.io  N  NaOH.  The  other  solutions  are  0.20  N  sodium  acetate  and 
0.20  N  acetic  acid.  Solutions  of  known  hydrogen  ion  concentration  are 
prepared  from  these  by  mixing  in  the  proportions  indicated  on  the 
chart,  the  abscissae  representing  parts  of  the  more  alkaline  or  less  acid 
constituent.  Thus  a  mixture  of  seven  volumes  of  the  sodium  acetate 
stock  solution  with  three  volumes  of  the  stock  acetic  acid  solution 


GASTRIC   ANALYSIS 


157 


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158 


PHYSIOLOGICAL    CHEMISTRY 


gives  a  mixture  with  an  hydrogen  ion  concentration  of  i  X  io~^ 
(exponent:  5.0).  The  mixtures  are  most  satisfactory  through  the 
ranges  where  the  hydrogen  ion  concentration  changes  most  gradually, 
that  is,  through  the  flatter  portions  of  the  curves. 

The  amounts  of  indicator  solutions  and  their  strengths  to  be  used 
in  the  determinations  of  hydrogen  ion  concentrations  in  10  c.c.  portions 
of  unknown  solution  are  indicated  below. 


I. 

Alizarin    yellow    R    (p- 
nitrobenzene-azo-salic- 

2. 

3- 

ylic  acid) 

Azolitmin  (litmus) 
Cochineal 

lo-s 

4- 
5- 

2,  5-dinitro-hydroquinone 
Mauvein 

5-2 
8-1 

6. 

Methyl  orange 

5-3 

7- 
8. 

9- 

Methyl  red 
Methyl  violet 
Neutral  red 

4-2 

8-1 

20-10 

10. 
II. 
12. 

p-Nitrophenol 
Phenolphthalein 
Rosolic  acid 

20-3 
20-3 
15-6 

13- 
14. 

IS- 

Thymolphthalein 
Tropaeolin  0 
Tropaeolin  00 

10-3 
10-5 
S-3 

16.  Tropaeolin  000 


INDICATOR  SOLUTIONS 

Drops  Preparation  of  solution 


o.  I  gram  to  1000  c.c.  water. 

Aqueous  solution. 

Alcoholic  solution. 

I  gram  to  1000  c.c.  alcohol. 

o.  5  gram  to  looo  c.c.  water. 

0.1    gram    recrystallized    salt    to    1000    c.c. 

water. 
Saturated  solution  in  50  per  cent,  alcohol. 
0.5  gram  to  1000  c.c.  water, 
o.  I  gram  in  500  c.c.  alcohol,  and  500  c.c.  water 
0.4  gram  to  60  c.c.  alcohol,  940  c.c.  water. 
0.5  gram  to  500  c.c.  alcohol,  500  c.c.  water, 
o .  4  gram  to  400  c.c.  alcohol,  600  c.c.  water. 
0.4  gram  to  500  c.c.  alcohol,  500  c.c.  water, 
o.  I  gram  to  1000  c.c.  water. 
Of  recrystallized  salt,  o.i  gram  to  1000  c.c. 

water. 
10-4  0.1  gram  to  1000  c.c.  water. 


Determination  of  Hydrogen  Ion  Concentration. — Introduce  10  c.c.  portions 
of  the  unknown  solution  into  a  series  of  test-tubes  of  similar  diameter  and  of 
clear  glass.  Test  first  with  Utmus  paper  which  changes  at  about  the  neutral 
point.  According  to  whether  the  reaction  is  acid  or  basic  to  litmus  test  other  indi- 
cators on  the  acid  side  such  as  p-nitrophenol,  methyl  orange  and  tropaeolin  00, 
or  on  the  basic  side  as  phenolphthalein.  Select  an  indicator  which  gives  with  the 
solution  neither  its  maximum  acid  or  maximum  basic  color.  Note  from  the  chart 
through  what  range  this  indicator  exhibits  its  characteristic  change  of  color. 
Then  to  10  c.c.  portions  of  standard  solutions  of  known  hydrogen  ion  concentra- 
tion (furnished  by  the  instructor),  which  cover  approximately  the  same  range  as 
the  indicator  add  exactly  the  same  number  of  drops  of  indicator  solution  as  was 
added  to  the  standard.  Compare  colors  of  unknown  and  standards  until  one  is 
found  which  matches  and  which  consequently  possesses  the  same  hydrogen  ion 
concentration.  If  the  unknown  is  so  strongly  acid  or  basic  that  none  of  the  indi- 
cators mentioned  can  be  used  directly  it  will  be  necessary  to  dilute  it  with  10  or  a 
greater  number  of  volumes  of  water  before  testing  further. 

In  case  the  unknown  solution  is  slightly  colored  the  standards  should  like- 
wise be  brought  to  the  same  tint  by  the  addition  of  some  coloring  agent  as  Bis- 
marck brown,  methyl  orange,  methyl  violet,  etc.,  before  making  the  comparison. 

For  appUcations  of  the  indicator  method  for  the  determination  of  hydrogen  ion 
concentration  to  biological  fluids  see  chapters  on  the  quantitative  analysis  of 
blood  (XVI)  and  urine  (XXVI). 

Comparison  of  H  Ion  Concentration  and  Titratable  Acidity. — i.  Determine 


GASTRIC   ANALYSIS  1 59 

colorimetrically  the  H  ion  concentration  of  an  N/ioo  solution  of  hydrochloric  acid 
using  tropaeolin  00  as  an  indicator  and  of  an  N/ 100  acetic  acid  using  methyl 
orange  as  an  indicator.  Note  the  great  difference  between  the  true  acidities  of 
the  two  solutions. 

Titrate  10  c.c.  portions  of  N  100  hydrochloric  acid  and  of  N/ioo  acetic  acid 
with  N/ioo  KOH  using  phenolphthalein  as  an  indicator.  Note  that  identical 
results  are  obtained  for  the  titratable  acidities  of  the  two. 

2.  Determine  colorimetrically  the  H  ion  concentration  of  an  N;ioo  KOH 
solution  using  tropagolin  O  as  an  indicator,  and  of  an  N/ioo  ammonia  solution 
using  phenolphthalein  as  an  indicator.  Note  the  results  and  then  titrate  10  c.c. 
portions  of  both  solutions  with  N/ioo  HCl  using  alizarin  as  an  indicator. 

3.  Mix  equal  portions  of  M  15  potassium  dihydrogen  phosphate  and  M  15 
disodium  phosphate  (see  chart).  Note  that  the  mixture  is  practically  neutral  to 
litiDius.  Titrate  one  10  c.c.  portion  of  this  mixture  with  N/io  KOH,  using  phe- 
nolphthalein as  an  indicator.  Titrate  another  portion  with  N/'io  HCl  solution, 
using  methyl  orange  as  an  indicator. 

4.  Mix  equal  volumes  of  N/5  sodiiun  acetate  solution  and  N/5  acetic  acid. 
Note  that  the  mixture  is  acid  to  litmus.  Titrate  one  10  c.c.  portion  with  N  10 
HCl  using  tropaeolin  00  as  an  indicator.  Titrate  another  portion  with  N  10 
KOH  using  phenolphthalein  as  an  indicator. 

THE  FRACTIONAL  METHOD  OF  GASTRIC  ANALYSIS 

Procedure  in  Gastric  Analysis  by  the  Fractional  Method 

1.  Introduction  of  the  stomach  tube  (see  pages  159  and  160). 

2.  Removal  of  the  residuum  (see  pages  160  and  161). 

3.  Feeding  the  test  meal  (see  page  161). 

4.  Feeding  the  retention  meal  (in  special  cases),  see  page  i6i. 

5.  Removing  samples  of  stomach  contents  for  analysis  (see  page  i6i). 

6.  Examination  of  the  samples  for: 

(a)  Total  acidity  (see  page  162). 

(b)  Free  acidity  (see  page  164). 

(c)  Pepsin  (see  page  165). 

(d)  Tripsin  (not  a  routine  procedure),  see  page  169. 

(e)  Lactic  acid  (see  page  170). 
(/)  Occult  blood  (see  page  171). 
{g)  Bile  (see  page  171). 

(/;)  Microscopical  constituents  (sec  page  173). 
I.  Introduction  of  the  Stomach  Tube. — Whereas  the  large  tube  is 
directly  inserted  by  propulsion,  the  Rehfuss  tube  is  swallowed  in  the 
natural  manner  and  aided  by  gravity.  The  tube  may  be  passed  in  one 
of  three  ways,  i.e.:  (i)  lubricated;  (2)  with  aid  of  lluid;  (3)  after  throat  is 
cocainized.  When  passed  by  the  first  method  the  tip  of  the  tube,  after 
thorough  lubrication  with  glycerol  or  liquid  petrolatum,  is  seized  between 


l6o  PHYSIOLOGICAL    CHEMISTRY 

the  thumb  and  forefinger  and  placed  on  the  tongue.  Then  with  the 
aid  of  the  forefinger  the  tip  is  pushed  forward  until  it  reaches  the  root  of 
the  tongue  and  is  engaged  in  the  oropharynx.  Then  the  patient  is 
encouraged  to  swallow  persistently  while  the  tube  is  slowly  fed  into  the 
mouth.  After  slight  discomfort  in  the  pharynx  and  its  passage  past  the 
level  of  the  cricoid  cartilage,  practically  no  discomfort  is  felt.  This 
method  is  used  when  it  is  essential  that  the  pure  gastric  secretion  or 
residuum  be  obtained.  Ordinarily,  however,  it  is  much  easier  to  swallow 
the  tube  by  the  second  method.  This  method  consists  in  placing 
the  tip  in  the  oropharynx  and  then  giving  the  patient  a  measured 
quantity  of  water  or  tea  to  swallow.  The  movements  induced  by  the 
swallowing  carry  the  tube  rapidly  to  the  stomach  with  a  minimum  of 
discomfort.  When  an  Ewald  meal  (see  below)  is  given,  part  of  the  tea 
can  be  reserved  for  swallowing  the  tube.  This  procedure  makes  it 
scarcely  more  arduous  than  the  swallowing  of  food.  Should  the  patient, 
however,  be  extremely  neurotic  or  the  unfortunate  possessor  of  marked 
pharyngeal  hyperesthesia,  cocain  hydrochloride  in  2  per  cent  aqueous 
solution  can  be  applied  to  the  throat  rendering  the  passage  of  the  tube 
practically  insensitive.  When  the  tube  has  entered  the  stomach,  as- 
piration of  the  material  shows  the  characteristic  gastric  contents. 
Should  the  tip  remain  in  the  esophagus  through  transient  cardiospasm 
or  other  cause,  aspiration  results  in  the  removal  of  only  a  very  small 
specimen  having  all  the  characteristics  of  the  pharyngeal  and  esoph- 
ageal secretions. 

2.  Removal  of  Residuum. — If  the  so-called  "empty"  stomach  is 
examined  in  the  morning  before  any  food  or  drink  has  been  taken  it 
will  be  found  to  contain  considerable  material.  This  is  termed  res- 
iduum. Before  a  test  meal  is  introduced  into  the  stomach,  this  organ 
should  be  emptied.  If  this  is  not  done  we  cannot  consider  the  samples 
withdrawn  after  the  test  meal  is  eaten  as  representing  the  secretory 
activity  of  the  gastric  cells  under  the  influence  of  the  stimulation  of  the 
test  meal.  It  has  been  generally  recognized,  clinically,  that  a  residuum 
above  20  c.c.  is  pathological.^  Such  a  volume  has  been  considered  as 
indicative  of  hypersecretion,  and  this  in  turn  in  many  cases  indicates  an 
organic  lesion.  The  observations  indicating  that  a  residuum  of  over 
20  c.c.  was  pathological,  were  made  upon  residuums  removed  by  means 
of  the  old  type  of  stomach  tube  which  does  not  completely  empty  the 
stomach.-     When  the  residuum  is  completely  removed  by  means  of 

*  Loeper:  Leqons  de  pathologie  digestive,  191 2,  Series  2,  pp.  17-19. 

Zweig:  Magen-  und  Darmkrankheiten,  p.  459. 

Kemp:  Diseases  of  the  Stomach,  Intestines  and  Pancreas,  191 2,  p.  133. 

Wolff:  Taschenbuch  der  Magen-  und  Darmkrankheiten,  p.  22. 
^  Harmer  and  Dodd:  Loc.  cit. 


GASTRIC    ANALYSIS  l6l 

the  Rehfuss  tube  it  has  been  demonstrated  that  the  normal  residuum 
is  practically  always  over  20  c.c.  and  that  the  average  is  about  50  c.c.^ 
The  normal  residuum  has  been  found  to  possess  all  the  qualities  of  a 
physiologically  active  gastric  juice  with  an  average  total  acidity  of  30 
and  an  average  free  acidity  of  18.5.  The  residuum  is  often  colored  by 
bile.  This  is  particularly  true  if  the  fluid  has  a  relatively  high  acidity. 
Trypsin  is  also  generally  present.  These  findings  indicate  regurgita- 
tion (see  page  151).  A  residuum  of  large  volume  possessing  a  total 
acidity  value  of  70  or  over  may  indicate  ulcer. 

Analysis  of  Residuum. — Remove  the  residuum  as  directed  under  (^5),  below, 
and  analyze  the  fluid  according  to  methods  outlined  on  page  162. 

3.  The  Test  Meal. — Before  making  an  analysis  of  the  stomach 
contents  it  is  customary  to  introduce  something  into  the  stomach  which 
will  stimulate  the  gastric  cells.  The  response  to  this  stimulation  is 
then  measured  clinically  by  the  determination  of  total  acidity,  free 
acidity  and  pepsin  in  the  stomach  contents.  ]\Iany  forms  of  test  meal 
have  been  used. 

The  test  meal  most  widely  employed  is  the  Ewald  test  meal.  This  consists 
of  2  pieces  (35  grams)  of  toast  and  8  ounces  (250  c.c.)  of  tea. 

Inasmuch  as  it  was  demonstrated  in  the  author's  laboratory^  that 
water  gave  a  similar  gastric  stimulation  to  that  produced  by  the  Ewald 
meal  it  was  suggested  that  a  simple  water  meal  might  be  substituted 
for  the  Ewald  meal.  This  water  meal  also  has  the  added  advantage 
of  enabling  one  to  determine  the  presence  of  food  rests  and  to  test  more 
accurately  for  lactic  acid,  blood  and  bile. 

4.  The  Retention  Meal. — In  order  to  obtain  more  information 
regarding  gastric  motility  than  is  furnished  by  the  ordinary  test  meal 
described  above  the  patient  may  be  fed  a  so-called  retention  meal.  This 
meal  is  fed  in  place  of  the  regular  evening  meal  and  contains  substances 
readily  detected.  In  the  morning  before  breakfast  (7-8  a.  m.)  remove 
the  stomach  contents  (residuum,  see  page  1 60)  by  aspiration  and  examine 
for  food  rests.  The  normal  stomach  should  give  no  eA-idences  of  food 
retention.  A  satisfactory  retention  meal  consists  of  4  ounces  each  of 
boiled  string  beans  and  rice.^  Diets  containing  prunes,  raspberry  mar- 
malade, lycopodium  powder,  etc.,  have  also  been  employed.  In  many 
instances  an  ordinary  mixed  diet  \\\\\  serve  the  purpose. 

5.  Removal  of  Samples  for  Analysis. — At  intervals  of  exactly  15 
minutes  from  the  time  the  test  meal  is  eaten  until  the  stomach  is  empty 

^  Rehfuss,  Bergeim  and  Hawk:  Jour.  Am.  Med.  Ass'n,  63,  11,  1914. 

Fowler,  Rehfuss  and  Hawk:  Jour.  Am.  Med.  Ass'n,  65,  1021,  1915. 
-  Bergeim,  Rehfuss,  and  Hawk:  Jour.  Biol.  Cfiem.,  19,  345,  1914- 

Rehfuss,  Bergeim  and  Hawk:  Jour.  Am.  Med.  Ass'n,  63,  11,  1914. 
'  Myers  and  Fine:  Essentials  of  Pathological  Chcmislry,  1913. 
II 


l62  PHYSIOLOGICAL    CHEMISTRY 

5-6  c.c.  samples  of  gastric  contents  are  withdrawn  from  the  stomach 
by  means  of  aspiration. 

In  the  removal  of  samples  from  the  stomach,  it  is  essential  that  very- 
little  traction  be  employed.  To  completely  empty  the  stomach,  aspira- 
tion is  practised  in  four  positions :  (a)  on  the  back ;  (b)  on  the  stomach ; 
(c)  on  right  side,  {(t)  on  left  side.  This  results  in  complete  evacuation 
of  the  stomach.  Three  tests  may  be  employed  to  determine  whether 
the  stomach  is  empty:  (i)  No  more  material  can  be  aspirated  in  any 
position;  (2)  injection  of  air  and  auscultation  over  the  stomach  with  a 
stethoscope  reveals  a  sticky  rale  and  not  a  series  of  gurgling  rales 
such  as  is  heard  when  there  is  material  in  the  stomach;  (3)  lavage  or 
irrigation  through  the  tube  which  shows  the  absence  of  all  food  in  the 
stomach. 

6.  Examination  of  the  Samples.- — The  old  methods  of  gastric  analy- 
sis involved  the  collection  (by  analysis  and  calculation)  of  data  regard- 
ing several  types  of  acidity  (see  Topfer's  method,  page  174).  The 
modern  tendency  among  clinicians  is  to  lay  particular  emphasis  upon 
the  values  for  total  acidity  and  free  acidity.  The  determination  of  the 
peptic  activity  is  also  important  as  well  as  the  demonstration  of  the 
presence  or  absence  of  occult  blood,  lactic  acid,  mucus,  food  rests,  etc. 

Procedixre. — Strain  each  sample  through  a  fine-mesh  cheese  cloth.  ^  Examine 
the  residue  for  mucus,  blood  and  food  rests.  Use  the  strained  stomach  contents 
for  the  determination  of  total  acidity,  free  acidity  and  peptic  activity  by  methods 
which  follow. 

(a)  Determination  of  Total  Acidity. — Pririciple. — The  indicator 
used  is  phenolphthalein.  Since  the  indicator  reacts  with  mineral 
acid,  organic  acid,  combined  acid  and  acid  salts  the  values  obtained 
represent  the  total  acidity  of  the  solution. 

Procedure.— Measure  i  c.c.  of  the  strained  stomach  contents  by  means  of  an 
Ostwald  pipette  and  introduce  it  into  a  low-form  60  c.c.  porcelain  evaporating 
dish.  Dilute  with  15  c.c.  of  distilled  water.  Add  2  drops  of  a  i  per  cent  alcoholic 
solution  of  phenolphthalein  and  titrate  with  N/ioo  sodium  hydroxide  until  a 
faint  pink  color  is  obtained  and  persists  for  about  two  minutes.  ^  Take  the  burette 
reading  and  calculate  the  total  acidity. 

*  The  examination  for  microscopical  constituents  (see  {h)  p.  173)  should  be  made  on  the 
original  (unstrained)  gastric  contents.  Tests  for  occult  blood  may  be  made  on  the  sedi- 
ment if  desired. 

"^  Procedure  for  Serial  Titrations. — When  a  series  of  titrations  are  to  be  made  the  following 
procedure  may  be  used:  Arrange  the  numbered  evaporating  dishes  in  rows  on  a  tray.  In- 
troduce I  c.c.  of  the  proper  sample  into  each  dish,  dilute  with  10  c.c.  of  water  and  add  the 
indicator.  Add  the  N/ioo  NaOH  to  contents  of  dish  No.  i  at  a  definite  rate  until  a  point  is 
reached  at  which  a  faint  pink  color  is  obtained,  as  described  above.  Return  dish  No.  i  to 
its  place  in  the  tray  and  place  dish  No.  2  under  the  burette.  Take  the  burette  reading  of 
No.  I.  Then  titrate  No.  2  in  the  same  way.  Continue  the  series.  This  procedure  has  the 
advantage  of  being  speedy  and  accurate.  There  is  a  slight  error  made  by  the  rapid  addition 
of  the  NaOH  but  it  is  uniform  and  the  results  (titrations)  are  therefore  comparable. 


GASTRIC    ANALYSIS 


163 


Calculation. — Note  the  number  of  cubic  centimeters  of  N/ 100  NaOH  required 
to  neutralize  i  c.c.  of  stomach  contents,  and  multiply  it  by  10  to  obtain  the  number 
of  cubic  centimeters  N/io  NaOH  necessary  to  neutralize  100  c.c.  of  stomach 
contents.  This  is  the  method  of  calculation  most  widely  used.  For  other  forms 
of  expressing  total  acidity  see  page  174.  Plot  your  results  in  a  form  similar  to 
those  shown  in  Figs.  45  and  46. 

Curves  Obtained  by  the  Fractional  Method. — When  an  Ewald  test  meal 
is  given  to  normal  individuals  a  curve  such  as  indicated  below  is  usu- 
ally obtained.  The  curve  may  vary  within  certain  limits  depending  on 
individual  idiosyncrasies,  but  is  usually  found  to  follow  the  curve 
depicted,  and  the  meal  normally  leaves  the  stomach  in  two  and  one- 


iOO 


20  40  60  60  100  120 

Fig.  45. — Acidity  Curves  of  XoRiiAL  Human  Stomach. 


half  hours.  Pathologically  every  variation  occurs,  both  in  time  of 
evacuation  as  well  as  the  character  of  the  curve  and  the  quantity  of  the 
secretion  elaborated.  Fig.  41  represents  some  of  the  possibilities  of 
pathological  cases,  but  a  consideration  of  their  interpretation  is  outside 
the  purpose  of  the  present  volume.  It  will  be  evident,  however,  from 
a  consideration  of  the  figure  that  the  cycle  of  gastric  digestion  is  a  con- 
stantly changing  one,  and  no  information  concerning  the  trend  of 
digestion  can  be  obtained  by  an  examination  of  only  a  single  stage  of 
digestion.  Marked  changes  may  precede  or  follow  that  stage  and  the 
possibilities  suggested  in  Fig.  41  are  all  observed  clinically  and  are  of 
varying  significance.  Typical  curves  from  cases  of  hyperacidity, 
gastric  carcinoma  and  achylia  are  shown  in  Figs.  46,  47  and  48 
respectively. 


164 


PHYSIOLOGICAL   CHEMISTRY 


(b)  Determination  of  Free  Acidity. — The  reagent  most  widely  used, 
clinically,  for  the  determination  of  free  hydrochloric  acid  in  stomach 

100 

Total  acidih| 


x: 
o 

to 


o 


Free  acidity 


'A    V/z    IV4.    2  hours 

Fig.  46. — Acidity  Curves  From  a  Case  of  Hyperacidity. 

contents  is  Topfer's  reagent  (see  page  175).     It  has  been  found,  however, 
that  this  reagent  gives  rather  inaccurate  results  due  to  the  uncertain 


c 

I 

1 

1120 
960 

800^ 

u 

640§ 
to 
z 

480^ 

320  80 

60 

160  40 

1 

1 

i 

(7a. 
Cat 

<itric 
cinon 

a 

i 

i 

•§' 

1 

4' 

/ 

/ 

/ 

y' 

y' 

^ 

^^ 

ilac 

^^ 

y' 

^_,..^-' 



I'ic 

,^'' 

Fre 

Fig.  47. — Acidity  and  Protein  Curves  in  Gastric  Carcinoma. 
Jour.  Am.  Med.  Ass'n,  64,  1737,  1915-) 


(Clarke  and  Rehfuss: 


end  point.     For  this  reason  we  have  employed  Sahli's  reagent.^     This 
reagent  contains  KI  and  KIO3  and  liberates  iodine  in  the  presence  of 

^  A  mixture  of  equal  parts  of  a  48  per  cent  solution  of  potassium  iodide  and  an  8  per  cent 
solution  of  potassium  iodate. 


GASTRIC    ANALYSIS 


165 


free  hydrochloric  acid.  The  liberated  iodine  is  titrated  by  thiosulphate 
using  starch  as  an  indicator.  It  gives  values  similar  to  Topfer's  re- 
agent in  average  acidities.^  Acidities  other  than  free  hydrochloric  re- 
act to  a  certain  extent  with  Sahli's  reagent. 

Procedure. — Measure  i  c.c.  of  the  strained  stomach  contents  by  means  of  an 
Ostwald  pipette  and  introduce  it  into  a  60  c.c.  porcelain  evaporating  dish.  Dilute 
with  ID  c.c.  of  distilled  water,  and  add  i  c.c.  of  Sahli's  reagent  (a.  mixture  of 
equal  parts  of  48  per  cent  KI  and  8  per  cent  KIO3).  Allow  the  stomach  contents 
thus  treated  to  stand  for  five  minutes  and  then  titrate  with  N/ioo  sodium  thio- 
sulphate imtil  only  a  faint  yellow  color  remains.  Now  add  5-10  drops  of  a  i 
per  cent  solution  of  soluble  starch  and  continue  the  titration  until  the  blue  color 
disappears.  In  serial  titrations  the  same  procedure  may  be  employed  as  de- 
scribed on  page  162,  note  2. 

Calculation. — Note  the  number  of  cubic  centimeters  of  N/ioo  sodium  thio- 
sulphate required  to  titrate  i  c.c.  of  stomach  contents  to  the  total  disappearance 
of  blue  color  in  the  presence  of  starch.  Inasmuch  as  N/ioo  thiosulphate  is 
equivalent  to  N/ioo  alkaU,  this  value  indicates  the  number  of  cubic  centimeters 
of  N/ioo  sodiimi  hydroxide  necessary  to  neutralize  the  free  hydrochloric  acid  in 


"p  ^0 

80 
40 

, 

-^ 

. — '   " 

Hours 

V4 

V2 

3/4 

I        IV4 

V/z 

1%. 

Fig.  48. — Total  Acidity  and  Protein  Curves  in  Benign  Achylia  (Solid  Line 
Represents  Acidity).     {Clarke  and  Rehfuss:  Jour.  Am.  Med.  Ass'n.,  64,  1737,  1915.) 

I  c.c.  of  the  stomach  contents.  Multiply  the  value  by  10  to  obtain  the  number  of 
cubic  centimeters  of  N/io  NaOH  necessary  to  neutrahze  100  c.c.  of  stomach 
contents.  This  is  the  method  of  calculation  most  widely  used.  For  other  forms 
of  expressing  free  acidity  see  page  174.  Plot  your  results  in  a  curve  similar 
to  those  shown  in  Figs.  43,  45,  and  46,  pages  150,  163  and  164. 

(c)  Determination  of  Peptic  Activity. — (i)  Method  of  Mett-  as 
Modified  by  Nirenstein  and  Scln&.^—Frinicplc. — Small  glass  tubes 
filled  with  coagulated  egg  albumin  are  introduced  into  tlie  solution  to 
be  tested,  and  kept  for  a  definite  length  of  time  in  the  incubator.  The 
protein  column  is  digested  at  both  ends  of  the  tube  to  an  extent  depend- 
ing upon  the  amount  of  pepsin  present.  The  method  is  not  strictly 
accurate  but  is  the  most  satisfactory  for  clinical  purposes  on  account 
of  its  simpUcity.  Nirenstein  and  Schiff  showed  that  human  gastric 
juice  contained  inhibiting  substances  the  effect  of  which  is  overcome  by 
the  dilution  recommended. 

1  Fowler,  Bergeim  and  Hawk:    Unpublished  data. 

2  Mett:  Arch.  f.  Anal.  u.  Physiol.,  1804.  68. 

^Nirenstein  and  Schiff:  Arch.  f.    Vcrdauungskraukhcilen,  8,   559,   1902. 


1 66  PHYSIOLOGICAL   CHEMISTRY 

Procedure. — Introduce  into  a  small  Erlemneyer  flask  i  c.c.  of  gastric  juice 
and  15  c.c.  of  N/20  HCl  (  =  0.18  per  cent  HCl).  Add  two  Mett  tubes  prepared 
as  indicated  below,  stopper  the  flask  to  prevent  evaporation  and  place  in  an  in- 
cubator at  37°C.  for  24  hours.  By  means  of  a  low  power  microscope  and  a  milli- 
meter scale  (graduated  to  half  millimeters)  determine  accurately  the  length  of 
the  column  of  albumin  digested  at  each  end  of  the  tubes.  It  is  well  to  run  the 
determination  in  dupUcate  in  which  case  the  result  is  the  average  of  the  eight 
figures  obtained.  Ordinarily  from  2-4  mm.  of  albumin  are  digested  by  normal 
human  gastric  juice. 

Calculation. — The  peptic  power  is  expressed  as  the  square  of  the  nxmiber  of 
millimeters  of  albumin  digested.  This  is  based  on  the  Schiitz-Borissow  law  that 
the  amount  of  proteolytic  enzyme  present  in  a  digestion  mixture  is  proportional 
to  the  square  of  the  number  of  millimeters  of  albumin  digested.  Therefore  a 
gastric  juice  which  digests  2  mm.  of  albumin  contains  four  times  as  much  pepsin 
as  one  which  digests  only  i  mm.  of  albvunin. 

Example. — If  the  microscopic  reading  gives  on  an  average  2.2  mm.  of  albumin 
digested  the  pepsin  value  for  the  diluted  juice  would  be  2.2 -  =  4.84,  and  for  the 
pure  undiluted  juice,  4.84X16  =  77.44. 

Preparation  of  Mett  Tubes  {Christiansen's  Method).^ — The  liquid  portions  of 
the  whites  of  several  eggs  are  mixed  and  strained  through  cheese  cloth.  The  mix- 
ture should  be  homogeneous  and  free  from  air  bubbles.  A  number  of  thin-walled 
glass  tubes  of  1-2  mm.  internal  diameter  are  thoroughly  cleaned  and  dried  and  cut 
into  lengths  of  about  10  inches.  These  are  sucked  full  of  the  egg-white  and  kept 
in  a  horizontal  position.  Into  a  large  evaporating  dish  or  basin  5-10  liters  of  water 
are  introduced  and  heated  to  boiling.  The  vessel  is  then  removed  from  the  fire 
and  stirred  with  a  thermometer  until  the  temperature  sinks  to  exactly  85°C.  The 
tubes  filled  with  egg-white  are  immediately  introduced  and  left  in  the  water  until 
it  has  cooled.  The  tubes  thus  prepared  are  soft  boiled,  more  easUy  digested  than 
hard  boiled  tubes,  and  free  from  air  bubbles.  The  ends  are  sealed  by  dipping  in 
melted  parafifin  or  sealing  wax  (preferably  the  latter),  and  the  tubes  can  be  kept  thus 
for  a  long  time.  When  ready  for  use  mark  with  a  file  and  break  into  pieces  about 
^  inch  long.  After  cutting,  the  tubes  should  be  immediately  introduced  into  the 
digestion  mixture  or  may  be  kept  a  short  time  under  water.  Tubes  whose  ends  are 
not  squarely  broken  off  must  be  rejected. 

The  digestibility  of  different  egg-whites  varies  widely.  Hence  in  making  up 
a  new  set  of.  tubes  if  we  wish  our  results  to  be  comparable  these  tubes  must  be 
standardized  against  those  first  prepared.  This  may  be  done  by  running  simul- 
taneous tests  with  tubes  from  the  two  series,  using  the  same  gastric  juice  and  com- 
paring the  lengths  of  the  columns  digested  in  each  case.  Christiansen's  method  of 
preparing  tubes  of  the  same  digestibility  is  to  be  preferred.  He  proceeds  as  in 
the  original  preparation  of  the  tubes  except  that  as  the  water  cools  from  90°-8o°C. 
a  single  tube  containing  the  new  egg-white  is  dropped  in  at  each  degree  change  of 
temperature,  that  is  at  90°,  89°,  etc.  Pieces  of  each  of  these  tubes  as  well  as  of  the 
original  standard  tubes  are  then  allowed  to  digest  simultaneously  in  portions  of  the 
same  gastric  juice.  One  of  these  tubes  should  show  a  digestibility  equal  to  that 
of  the  standard  tubes.  For  example  the  tube  coagulated  at  88°C.  may  show  the 
proper  digestibility.  Then  the  new  series  of  tubes  should  be  made  in  the  same  man- 
ner as  this  one,  that  is  introduced  at  88°C.  The  tubes  thus  prepared  should  be 
again  checked  up  with  the  standard  to  see  that  no  mistake  has  been  made. 
^  Christiansen:  Biochem.  Zeit.,  46,  257,  1912. 


GASTRIC   ANALYSIS  1 67 

(2)  Fuld  and  Levison's  Method. — This  test  is  founded  upon  the  fact,  shown  by 
Osborne,  that  edestin  when  brought  into  solution  in  dilute  acid  will  change  in  its 
solubility,  due  to  the  contact  with  the  acid,  and  that  a  protean  called  edes tan, v>'h\ch 
is  insoluble  in  neutral  fluid,  will  be  formed.  The  procedure  is  as  follows:  Dilute 
the  gastric  juice  under  examination  with  20  volumes  of  water  and  introduce  gradu- 
ally decreasing  volumes  of  the  diluted  juice  into  a  series^  of  narrow  test-tubes  about 
I  cm.  in  diameter.  The  measurements  of  gastric  juice  may  conveniently  be  made 
with  a  I  c.c.  pipette  which  is  accurately  graduated  in  Hoo  c-c.  Into  the  first  tube 
in  the  series  may  be  introduced  i  c.c.  of  gastric  juice,  and  the  tubes  which  follow  in 
the  series  may  receive  volumes  which  differ,  in  each  instance,  from  the  volume  intro- 
duced into  the  preceding  tube  by  Koo;  Ho;  Mo;  or  Ho  o^  ^  cubic  centimeter. 
Now  rapidly  introduce  into  each  tube  the  same  volume  {e.g.,  2  c.c.)  of  a  i  :  1000 
solution  of  edestin-  and  place  the  tubes  at  4o°C.  for  one-half  hour.  At  the  end  of 
this  time  stratify  ammonium  hydroxide  upon  the  contents  of  each  tube,-'  place  the 
tubes  in  position  before  a  black  background  and  examine  them  carefully.  The 
ammonium  hydroxide,  by  diffusing  into  the  acid  fluid,  forms  a  neutral  zone  and  in 
this  zone  will  be  precipitated  any  undigested  edestan  which  is  present.  Select  the 
tube  in  the  series  which  contains  the  least  amount  of  gastric  juice  and  which  exhibits 
no  ring,  signifying  that  the  edestan  has  been  completely  digested,  and  calculate  the 
peptic  activity  of  the  gastric  juice  under  examination  on  the  basis  of  the  volume  of 
gastric  juice  used  in  this  particular  tube. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  edestin  solution  used 
by  the  dilution  to  which  the  gastric  juice  was  originally  subjected  and  divide  the 
volume  of  gastric  juice  necessary  to  completely  digest  the  edestan  by  this  product. 
For  example,  if  2  c.c.  of  the  edestin  solution  was  completely  digested  by  0.25  c.c. 
of  a  I  :  20  gastric  juice  we  would  have  the  following  expression :  o.25-i-(2oX2)  or 
I  :  160.  This  peptic  activity  may  be  expressed  in  several  ways,  e.g.,  (a)  i  :  160  pep- 
sin; (b)  160  pepsin  content;  (c)  160  parts. 

(3)  Rose's  Modification^  of  the  Jacoby-Solms  Method.^ — Dissolve  0.25  gram  of 
the  globulin  of  the  ordinary  garden  pea,^  Pisiim  sativum,  in  100  c.c.  of  10  per  cent 

1  The  longer  the  series,  the  more  accurate  the  deductions  which  maj"  be  drawn. 

2  This  edestin  should  be  prepared  in  the  usual  way  (seep.  109),  and  brought  into  solution 
in  a  dilute  hydrochloric  acid  of  approximately  the  same  strength  as  that  which  occurs  nor- 
mally in  the  human  stomach.  This  may  be  conveniently  made  by  adding  30  c.c.  of  X^  10 
hydrochloric  acid  to  70  c.c.  of  water.  Ordinarily  it  should  not  take  longer  than  one  minute 
to  introduce  the  edestin  solution  into  the  entire  series  of  tubes.  However,  if  the  edestin  is 
added  to  the  tubes  in  the  same  order  as  the  ammonium  hydroxide  is  afterward  stratified,  no 
appreciable  error  is  introduced. 

^  Making  the  stratification  in  the  same  order  as  the  edestin  solution  was  added. 

*  Rose:  Archives  of  Internal  Medicine,  5,  459,  1910. 

*  Solms:  Zeitschrifl  JUr  klinische  Medizin,  64,  159,  1907. 

'  The  globulin  may  be  prepared  as  follows:  "The  finelj'  ground  peas,  freed  as  much  as 
possible  from  the  outer  coating,  are  repeatedly  extracted  with  large  quantities  of  10  per  cent 
sodium  chloride  solution,  the  extracts  combined,  strained  through  fine  bolting-cloth,  and 
allowed  to  stand  over  night  in  large  cyhnders  to  deposit  insoluble  matter.  The  supernatant 
fluid  is  siphoned  off  and  saturated  with  ammonium  sulphate.  The  precipitate  of  albumin 
and  globuHn  is  filtered  off,  suspended  in  a  httle  water,  and  dialyzed  in  running  water  for 
three  days,  until  the  salt  has  been  removed,  and  the  albumins  have  been  dissolved.  The 
globulins  are  filtered  off  and  washed  two  or  three  times  to  remove  the  last  trace  of  albumins. 
To  purify  further,  the  precipitate  is  extracted  with  10  per  cent  sodium  chloride  solution,  and 
filtered  until  perfectly  clear.  The  resulting  solution  is  neutrahzed  to  litmus  paper  by  the 
cautious  addition  of  dilute  sodium  hydroxide,  and  again  dialyzed  in  running  water  for  three 
days  to  remove  the  salts  completely.  The  precipitated  globulins  are  then  filtered  off  and 
dried  on  a  water-bath  at  40°C.  During  the  entire  process  of  separation  the  proteins  should 
be  preserved  with  a  mixture  of  alcoholic  thymol  and  toluol."  This  dried  globulin  is  used  in 
the  clinical  procedure. 


1 68  PHYSIOLOGICAL   CHEMISTRY 

sodium  chloride  solution,  warming  slightly  if  necessary.^  FUter  and  introduce  i  c.c. 
of  the  clear  filtrate  into  each  of  a  series  of  six^  test-tubes  about  i  cm.  in  diameter. 
Introduce  into  each  tube  i  c.c.  of  0.6  per  cent  hydrochloric  acid  and  permit  a  period 
of  about  five  minutes  to  elapse  for  the  development  of  the  turbidity.  Make  a 
known  volume  of  the  gastric  juice  (5-10  c.c.  is  sufficient)  exactly  neutral  to  litmus 
paper  with  dilute  alkali;  and  record  the  volume  of  the  alkali  so  used.  If  acid 
metaprotein  precipitates,  filter  it  off;  if  there  is  no  precipitate  proceed  without 
filtration.  Dilute  the  clear  neutral  solution  with  a  known  quantity  of  distUled 
water  (usually  5  volumes)  making  proper  allowance  for  the  volume  of  alkali  used  in 
the  neutralization.  Boil  5-10  c.c.  of  the  diluted  juice,  filter  and  add  the  following 
decreasing  volumes  (c.c.)  to  the  series  of  six  tubes:  i.o,  0.9,  0.7,  0.5,  0.2,  0.0.  Make 
the  measurements  by  means  of  a  i  c.c.  pipette  graduated  in  o.oi  c.c.  Now  rapidly 
introduce  the  unboiled,  diluted  juice  in  the  following  increasing  volumes  (c.c.)  in 
order:  0.0,  o.i,  0.3,  0.5,  0.8,  1.0.  Each  tube  now  contains  a  total  volume  of  3  c.c. 
and  a  total  acidity  of  0.2  per  cent  hydrochloric  acid.  Shake  each  tube  thoroughly 
and  place  them  at  5o-52°C.  for  15  minutes  or  at  35-36°C.  for  one  hour.  Examine 
the  series  of  tubes  at  the  end  of  the  digestion  period  and  select  that  tube  which 
contains  the  smallest  quantity  of  gastric  juice  and  which  shows  no  turbidity.  The 
volume  of  the  juice  used  in  this  tube  is  taken  as  the  basis  for  the  calculation  of  the 
peptic  activity. 

Calculation. — The  peptic  activity  is  expressed  in  terms  of  i  c.c.  of  the  undiluted 
juice.  For  example,  if  it  requires  0.5  c.c.  of  the  diluted  juice  (five-fold  dilution)  to 
clear  up  the  turbidity  in  i  c.c.  of  the  globulin  solution  in  the  proper  experimental 
time  interval  (15  minutes  or  one  hour  according  to  temperature)  the  peptic  activity 
would  be  expressed  as  follows: 

(i-r-o.5)X5  =  io  (peptic  activity). 

According  to  this  scale  of  pepsin  units  10  may  be  considered  as  "normal"  peptic 
activity.  These  units  are  about  Ko  as  large  as  those  expressed  by  the  Jacoby- 
Solms  scale. 

Inasmuch  as  it  has  been  shown'  that  blood  serum  contains  an  antipepsin  it  is 
advisable  to  test  the  gastric  juice  for  blood  before  determining  its  proteolytic  power. 

(4)  Given's  Modification  of  Rose's  Method.^ — The  gastric  contents  are 
strained  through  cheese  cloth.  Two  c.c  are  measured  by  means  of  an  Ostwald  pipette 
into  a  25  c.c  stoppered  volumetric  cylinder,  and  dUuted  to  the  mark  with  dis- 
tUled water.  Into  each  of  seven  small  test-tubes  (i  X 10  cm.)  is  measured,  with 
an  Ostwald  pipette,  i  cc  of  a  0.25  per  cent  filtered  pea  globulin  in  10  per  cent 
sodium  chloride  solution.  To  each  tube  is  added  i  c.c  of  0.6  per  cent  hydro- 
chloric acid,  also  by  means  of  an  Ostwald  pipette.  The  tubes  are  allowed 
to  stand  about  five  minutes,  untU  the  maximum  turbidity  develops.  To  the 
first  five,  distilled  water  is  added  as  follows:  To  the  first,  0.9  c.c;  to  the  second, 
0.8  cc;  to  the  third,  0.7  c.c;  to  the  fourth,  0.6  c.c;  and  to  the  fifth,  0.2  c.c;  to 
the  sixth  and  seventh,  none.  Then  there  are  rapidly  added  to  each  test-tube 
the  following  amounts  of  the  diluted  (i  :i2.5)  gastric  juice;  to  thefirst,  o.i  c.c.;to 
the  second,  0.2  cc;  to  the  third,  0.3  c.c;  to  the  fourth,  0.5  c.c;  to  the  fifth,  0.8  cc; 

^  This  solution  may  be  preserved  at  least  two  months  under  toluene. 
^  A  longer  series  of  tubes  may  be  used  if  desired.     However,  experience  has  shown  that 
a  series  of  six  ordinarily  affords  sufficient  range  for  all  diagnostic  purposes. 
'Oguro:  Biochemische  Zeitschrift,  22,  266,  1909. 
*  Givens:  Hygienic  Lab.  Bull.  loi,  p.  71,  August,  1915. 


GASTRIC   ANALYSIS  1 69 

to  the  sixth,  i.o  c.c;  and  to  the  seventh,  i.o  c.c.  of  the  diluted  juice  boiled.  These 
measurements  can  be  accurately  made  with  a  i  c.c.  pipette  graduated  in  o.oi  c.c. 
All  tubes  are  then  immersed  for  15  minutes  in  a  water-bath  at  50°  to  S2°C.  At  the 
end  of  this  time,  the  tube  is  selected  which  is  clear  and  contains  the  least  amount  of 
diluted  gastric  juice.  Upon  this  basis,  the  peptic  activity  is  calculated  as  the  num- 
ber of  cubic  centimeters  of  0.25  per  cent  globulin  digested  by  i  c.c.  of  undiluted 
gastric  juice.  For  example,  if  tube  2  containing  0.3  c.c.  of  a  12.5  times  diluted 
juice  be  clear,  then  the  result  would  be  expressed: 

Peptic  activity=  (i-j-o.3)Xi2.5  =  4i.2. 

Ordinarily  this  scheme  of  seven  tubes  is  used,  though  it  is  not  a  rule.  If  the 
free  acidity  be  high,  sometimes  a  dilution  of  3^5  is  made.  The  number  of  tubes 
used  will  depend  upon  the  accuracy  desired. 

(d)  Determination  of  Tryptic  Activity. — Trypsin  is  not  a  gastric 
enzyme  but  occurs  in  the  pancreatic  juice  (see  page  i88).  In  case  of 
regurgitation  of  intestinal  contents  through  the  pylorus  trypsin  would 
be  passed  into  the  stomach.  This  regurgitation  is  doubtless  of  frequent 
occurrence  and  may  even  be  a  normal  mechanism  by  which  gastric 
acidity  is  regulated  (see  page  151).  Trypsin  is,  therefore,  generally 
present  in  the  contents  of  the  normal  human  stomach. 

Spencer's  Method.' — (a)  Prepare  five  reagent  tubes,  Nos.  i,  2,3,4,  and  5;  more 
if  desired. 

To  tubes  I  and  2  add  0.5  c.c.  of  gastric  contents  (filter  if  cloudy). 

(b)  To  tubes  2,  3,  4,  and  5  add  0.5  c.c.  of  distilled  water. 

(c)  From  tube  2  remove  0.5  c.c.  of  its  mixed  contents  and  add  to  tube  3.  Mix 
thoroughly  and  add  0.5  c.c.  from  tube  3  to  tube  4.     Repeat  for  tube  5. 

We  now  have  dilutions  of  gastric  contents  of  i,  3^,  3^,  3^,  and  3^6- 

(d)  To  each  tube  add  one  drop  of  phenolphthalein  solution  (phenolphthalein 
I  gram;  alcohol  (95  per  cent)  100  c.c);  then  add  drop  by  drop  a  2  per  cent  sodium 
bicarbonate  solution  until  a  light  pink  color  is  produced. 

(e)  To  tubes  i,  2,  3,  and  4  add  0.5  c.c.  of  casein  solution.  Tube  5  must  receive 
I  c.c.  of  casein  solution,  since  it  contains  i  c.c.  of  the  diluted  gastric  contents.  For 
the  casein  solution,  dissolve  0.4  gram  of  casein  in  40  cc.  of  N/io  NaOH.  Add  130 
c.c.  of  distilled  water,  then  30  c.c.  of  N/io  HCl.  This  leaves  the  solution  alkaline 
to  the  extent  of  10  c.c.  of  N/io  NaOH,  minus  about  3  cc  neutralized  by  the 
casein. 

(J)  Incubate  for  five  hours  at  4o°C. 

(g)  Precipitate  the  undigested  casein  by  dropwise  addition  of  a  solution  of  the 
following  composition:  glacial  acetic  acid  i  c.c,  alcohol  (95  per  cent)  50  c.c,  dis- 
tilled water  50  cc.  The  tubes  in  which  digestion  has  been  complete  remain  clear; 
others  become  turbid. 

(h)  The  tryptic  values  are  expressed  in  terms  of  dilution.  Thus,  complete 
digestion  in  tube  3  (a  dilution  of  Ji)  shows  four  times  the  tryptic  power  of  un- 
diluted gastric  juice;  taken  as  a  standard  as  i,  therefore,  its  tryptic  value  is  4. 

^Elaborated  by  Dr.  W.  H.  Spencer  {Jour.  Biol.  Chem.,  21,  165,  1915)  in  the  author's 
laboratory  for  the  specific  purpose  of  determining  trypsin  in  gastric  juice.  For  other  trjp- 
sin  methods  see  Chapter  X. 


lyo  PHYSIOLOGICAL    CHEMISTRY 

(z)  Controls  of  boiled  gastric  contents  plus  casein  solution,  and  of  distilled  water 
plus  casein  solution,  treated  as  above  stated,  must  show  no  digestion,  and  become 
turbid  on  addition  of  the  precipitating  solution. 

(e)  Detection  of  Lactic  Acid. — WTien  the  acidity  of  the  stomach 
contents  is  reduced  to  a  low  value  there  may  occur  considerable  fermen- 
tation of  carbohydrates  which  have  been  introduced  into  the  stomach 
in  the  ingested  food.  This  fermentation  yields  various  organic  acids 
among  which  lactic  acid  is  particularly  prominent.  It  is  important, 
therefore,  in  case  of  low  gastric  acidity  that  the  stomach  contents  be 
examined  for  lactic  acid. 

Tests.  I.  Ether -Ferric  Chloride  Test  (Strauss).— A  satisfactory 
deduction  regarding  the  presence  of  lactic  acid  can  only  be  made  by 
removing  the  lactic  acid  from  disturbing  factors  {e.g.,  hydrochloric  acid, 
protein  digestion  products,  etc.)  present  in  the  stomach  contents. 
Lactic  acid  may  be  extracted  from  the  stomach  contents  by  ether. 
The  following  technic  not  only  serves  to  detect  lactic  acid  but  also  gives 
an  approximate  idea  as  to  the  amount  of  the  acid  present. 

Procedure. — Introduce  5  c.c.  of  strained  stomach  contents  into  a  small  grad- 
uated separatory  funnel,  add  20  c.c.  of  ether  and  shake  the  mixture  thoroughly. 
Permit  the  ether  to  separate,  then  allow  all  the  fluid  to  run  out  of  the  separatory 
funnel  except  the  upper  5  c.c.  of  ether.  To  this  ether  extract  add  20  c.c.  distilled 
water  and  2  drops  of  a  10  per  cent  solution  of  ferric  chloride  and  shake  the  mix- 
ture gently.  A  slight  green  color  is  obtained  in  the  presence  of  0.05  per  cent  lac- 
tic acid  whereas  o.  i  per  cent  lactic  acid  yields  a  very  intense  yellowish -green  color. 

2.  Ferric  Chloride  Test  (Kelling). — Fill  a  test-tube  with  water,  add  1-2 
drops  of  a  10  per  cent  solution  of  ferric  chloride  and  mix  thoroughly  to  form  a 
liquid  which  is  very  faintly  colored.  Divide  the  solution  into  two  parts  and  keep 
one  part  as  a  control.  To  the  other  part  add  a  small  amount  of  the  strained 
gastric  contents  and  to  the  control  tube  add  a  similar  volimie  of  water.  Lactic 
acid  is  indicated  by  the  immediate  development  of  a  distinct  yellow  color  in  the 
tube  containing  the  gastric  contents. 

The  color  in  this  test  is  due  to  the  formation  of  ferric  lactate. 

3.  Uffelmann's  Reaction. — To  5  c.c.  of  Uffelmann's  reagent^  in  a  test-tube 
add  an  equal  volume  of  strained  gastric  juice.  A  canary  yellow  or  greenish- 
yellow  color  develops  if  lactic  acid  be  present  to  the  extent  of  o.oi  per  cent  or  over. 

Other  organic  acid  gives  a  similar  reaction.  Mineral  acids  such  as 
hydrochloric  acid  discharge  the  blue  coloration  leaving  a  colorless 
solution.  In  other  words,  the  color  of  the  reagent  is  weakened  in  the 
presence  of  an  acid  reaction. 

^  Uffelmann's  reagent  is  prepared  by  adding  ferric  chloride  solution  to  a  i  per  cent  solu- 
tion of  carbolic  acid  until  an  amethyst-blue  color  is  obtained,  due  in  part  to  the  formation  of 
a  ferric  salt  of  carbolic  acid  and  in  part  to  the  reduction  of  some  of  the  iron. 


GASTRIC   ANALYSIS  171 

4.  Hopkins'  Thiophene  Reaction.— Place  about  5  cc.  of  concentrated  sulphuric 
acid  in  a  test-tube  and  add  i  drop  of  a  saturated  solution  of  copper  sulphate.^ 
Introduce  a  few  drops  of  the  gastric  contents,  shake  the  tube  well,  and  immerse  it 
in  the  boiling  water  of  a  beaker-water-bath  for  one  or  two  minutes.  Now  remove 
the  tube,  cool  it  under  running  water,  add  2-3  drops  of  a  dilute  alcoholic  solution- 
of  thiophene,  C4H4S,  from  a  pipette,  replace  the  tube  in  the  beaker  and  carefully 
observe  any  color  change  which  may  occur.  Lactic  acid  is  indicated  by  the  ap- 
pearance of  a  bright  cherry-red  color  which  forms  rapidly.  This  color  may  be  made 
more  or  less  permanent  by  cooling  the  tube  as  soon  as  the  color  is  produced.  Ex- 
cess of  thiophene  produces  a  deep  yellow  or  brown  color  with  sulphuric  acid.  The 
test  is  not  wholly  specific  though  the  author  claims  it  to  be  more  so  than  Uffelmann's 
reaction. 

(f)  Detection  of  Occult  Blood.  ^~i.  Ortho-toli din  Test  (RuttanandHardisty)." 
— To  I  cc.  of  a  4  per  cent  glacial  acetic  acid  solution  of  o-tolidin4na  test-tube  add 
I  cc.  of  the  gastric  juice  under  examination  and  i  cc.  of  3  per  cent  hydrogen 
peroxide.  In  the  presence  of  blood  a  bluish  color  develops  (sometimes  rather 
slowly)  and  persists  for  some  time  (several  hours  in  some  instances). 

This  test  is  said  to  be  as  sensitive  for  the  detection  of  occult  blood  in 
feces  and  stomach  contents  as  is  the  benzidine  reaction.  It  is  also 
claimed  to  be  more  satisfactory  for  urine  than  any  other  blood  test. 
The  acetic  acid  solution  may  be  kept  for  one  month  with  no  reduction  in 
delicacy. 

2.  Benzidine  Reaction. — This  is  one  of  the  most  delicate  of  the  reactions  for  the 
detection  of  blood.  Different  benzidine  preparations  vary  greatly  in  their  sensi- 
tiveness, however.  Inasmuch  as  benzidine  solutions  change  readily  upon  contact 
with  Ught  it  is  essential  that  they  be  kept  in  a  dark  place.  The  test  is  per- 
formed as  follows :  To  a  saturated  solution  of  benzidine  in  alcohol  or  glacial  acetic 
acid  add  an  equal  volume  of  3  per  cent  hydrogen  peroxide  and  i  cc  of  the  gastric 
contents  under  examination.  If  the  mixture  is  not  already  acid  render  it  so  with 
acetic  acid,  and  note  the  appearance  of  a  blue  color.  A  control  test  should  be 
made  substituting  water  for  the  solution  under  examination. 

The  sensitiveness  of  the  benzidine  reaction  is  greater  when  applied 
to  aqueous  solutions  than  when  applied  to  the  urine.  According  to 
Ascarelli  the  benzidine  reaction  serves  to  detect  blood  when  present  in 
a  dilution  of  1:300,000.  (For  further  discussion  of  this  test  see 
chapter  on  Blood.) 

(g)  Detection  of  Bile  in  Stomach  Contents. — If  we  accept  Boldyreff's 
theory  as  to  the  automatic  regulation  of  gastric  acidity^  under  normal 

'  This  is  added  to  catalyze  the  oxidation  which  follows. 
-About  10-20  drops  in  100  cc.  af  95  per  cent  alcohol. 

'  These  tests  maybe  made  upon  the  strained  stomach  contents  or  upon  the  sohd  residue. 
*  Ruttan  and  Hardisty:  Canadian  Medicine  Ass'n  Journal,  Nov.,  1912;  also  Biochem. 
Bull.,  2,  225,  1913. 

5  XHov  yXH., 

/CeH^  — C6H4\ 

CH3/  \CH, 

•Boldyreff:  Quart.  Jour.  Exp.  Med.,  8,  i,  1914. 


172 


PHYSIOLOGICAL    CHEMISTRY 


conditions  by  the  regurgitation  of  alkaline  material  from  the  intestine, 
then  the  presence  of  bile  in  the  gastric  juice  does  not  possess  the  clinical 
significance  it  has  been  accorded.  However,  if  an  ordinary  Ewald  meal 
be  fed,  and  bile  in  any  considerable  quantity  be  found  throughout  the 
entire  course  of  digestion  it  may  indicate,  pathologically,  a  stenosis 
below  the  level  of  the  common  bile  duct.  Frequently  samples  of 
gastric  contents  are  encountered  which  are  uncolored  and  which  never- 
theless contain  bile.  It  is  also  true  that  bile  may  be  adsorbed  from 
stomach  contents  by  mucus  and  food  rests.  The  regulation  technic  for 
bile  testing  is  often  inadequate  to  demonstrate  the  presence  of   this 


Fig.  49. — Microscopical  Constituents  of  the  Gastric  Contents. 

A,  Starch  cells;  B,  yeast  cells;  C,  Oppler-Boas  bacilli;  D,  staphylococci;  E,  streptococci; 
F,  sarcinae;  G,  muscle  iibre;  H,  mucus;  /,  red  blood  cells;  /,  leucocytes;  K,  snail-like  mucus 
formations;  L,  squamous  epithelial  cell;  Af,  cellulose. 

fluid  in  gastric  contents.  The  following  procedure  based  upon  the 
oxidation  of  the  bilirubin  with  nitric  acid  forming  green  biliverdin  is 
delicate  and  easy  of  application. 


Procedure. — Saturate  10  c.c.  of  the  fluid  portion  of  the  stomach  contents  with 
powdered  ammonium  sulphate.  This  may  be  accomplished  by  shaking  for  one 
to  two  minutes.  It  generally  requires  about  i  inch  of  powdered  sulphate  in 
the  bottom  of  an  ordinary  test-tube  to  obtain  full  saturation.  When  the  fluid  is 
saturated  add  1-3  c.c.  of  acetone  and  thoroughly  mix  the  contents  of  the  tube  by 
inverting  the  tube  five  or  six  times.  (It  is  better  not  to  shake.)  Permit 
the  tube  to  stand  and  allow  the  acetone  to  rise  to  the  surface.    This  acetone  con- 


GASTRIC   ANALYSIS  1 73 

tains  the  bile  pigment  if  any  is  present  in  the  stomach  contents.  Allow  a  drop 
of  yellow  nitric  acid  to  flow  down  the  side  of  the  tube  and  note  the  green  color  in 
the  acetone. 

This  green  color  is  biliverdin  which  has  been  produced  from  the 
bilirubin  by  oxidation  with  nitric  acid.  If  too  much  acid  is  added 
the  green  color  will  be  oxidized  to  a  purple  or  red.  If  the  acetone  does 
not  rise  to  the  surface  promptly  the  liquid  has  not  been  completely 
saturated  with  ammonium  sulphate. 

If  the  stomach  contents  contains  large  amounts  of  bile  as  indicated 
by  a  deep  green  color  4-5  drops  of  the  fluid  may  be  diluted  with  10  c.c. 
water  and  the  above  test  applied. 

(h)  Microscopy  of  the  Gastric  Contents. — A  microscopical  exami- 
nation of  the  gastric  contents  is  a  routine  clinical  procedure. 

When  an  Ewald  meal  is  given  the  starch  granules  in  various  stages 
of  digestion  are  observed  together  with  epithelia  from  the  pharynx, 
esophagus,  and  occasionally  the  stomach.  Gastric  and  salivar>' 
mucus  are  seen  and  readily  recognized  by  their  ropy  appearance. 
Pathologically  various  bacteria  are  seen,  sarcinae,  Oppler-Boas  bacilli, 
streptococci,  leptothrix,  etc.  Retained  food  from  previous  meals  is 
readily  recognized  by  its  histological  appearance;  meat  fibers,  vegetable 
cells,  and  cellulose  may  all  occur  in  pathological  retention.  In  certain 
pathological  processes  such  as. ulcer  and  cancer,  red  blood  cells,  pus, 
and  even  the  cancer  cells  themselves  may  be  found.  For  illustrations 
of  the  microscopical  constituents  of  gastric  contents,  see  Fig.  49. 

Procedure.— Examine  a  drop  of  the  original  (mixed)  stomach  contents  un- 
stained under  the  low  and  high  powers  of  the  microscope.  Compare  your  find- 
ings with  the  microscopical  views  shown  in  Fig.  49. 

Wolff  Technic  for  the  Protein  Concentration  of  the  Gastric  Contents.' — 
Owing  to  the  diagnostic  importance  of  the  protein  concentration  of  the  gastric 
secretion,  a  short  note  of  this  test  is  given  here.  Under  normal  conditions  the 
protein  concentration  follows  that  of  acidity  rather  closely.  In  certain  cases,  how- 
ever, such  as  carcinoma  (Fig.  47),  there  is  an  actual  increase  in  the  protein  concen- 
tration of  the  gastric  juice  out  of  ail  proportion  to  the  acidity.  The  test  may  be 
made  as  follows:  The  regular  Ewald  test  meal  is  fed  and  specimens  of  the  gastric 
contents  are  obtained  at  is-minute  intervals  by  means  of  the  Rehfuss  tube.  One 
c.c.  of  the  filtered  juice  is  then  diluted  with  9  c.c.  of  water  representing  a  dilution 
of  I  :io;  5  c.c.  of  this  mixture  is  again  added  to  5  c.c.  of  water  and  a  dilution  of  i  :2o 
obtained;  this  is  again  repeated  using  5  c.c.  of  the  mi.xturc  last  obtained  and  5  c.c. 
of  distilled  water  and  the  dilutions  are  kept  up  until  a  scries  is  obtained  representing 
1 :  10,  1 :  20.  1 :  40,  i :  80,  i :  160,  i :  320,  and  if  necessarj^  i :  640  or  more.     They  are 

1  Wolff:  Magen-  und  Darmkrankh.,  Berlin,  1912,  p.  217;  also  Berl.  klin.  Woch.,  May  29, 
1911,  and  March  i8,  1912. 

Rolph:  Ued.  Rec,   19 13,  p.   848. 

Clarke  and  Rehfuss:  Jour.  Am.  Med.  Ass'n,  64,  1737,  191 5. 


174  PHYSIOLOGICAL   CHEMISTRY 

then  stratified  with  approximately  i  c.c.  of  Wolff's  reagent/  care  being  taken  that 
the  liquids  do  not  mix.  The  tubes  should  be  read  immediately  against  a  dark 
background  and  the  tube  giving  a  protein  ring  at  the  greatest  dilution  of  gastric 
juice  recorded.  A  glance  at  Fig.  47  will  show  a  pronounced  case  of  gastric  carci- 
noma. With  normal  acid  figures  the  protein  concentration  evolves  proportionally 
to  the  acidity.     A  case  of  achylia  is  shown  in  Fig.  48. 


Topfer's  Method  of  Gastric  Analysis 

This  method  is  much  less  elaborate  than  many  others  but  is  sufl&ciently  ac- 
curate for  ordinary  clinical  purposes.  The  method  embraces  the  volumetric  de- 
termination of  (i)  total  acidity,  (2)  free  acidity  {organic  and  inorganic),^  and  {3)  free 
hydrochloric  acid,  and  the  subsequent  calculation  of  (4)  combined  acidity  and  (5) 
acidity  due  to  organic  acids  and  acid  salts,  from  the  data  thus  obtained. 

Procedure. — Feed  the  Ewald  test  meal  as  directed  on  page  161.  At  the  end  of 
one  hour  remove  the  entire  stomach  contents  and  analyze  as  directed  below.  This 
method  of  procedure  is  less  accurate  than  the  Fractional  Method  (see  page  148). 
Measure  the  volume  of  the  gastric  contents,  strain  it  through  cheese  cloth  and  intro- 
duce 10  cc  of  the  strained  material  into  each  of  three  small  beakers  or  porcelain 
dishes^  Label  the  vessels  A,  B,  and  C,  respectively,  and  proceed  with  the  analysis 
according  to  the  directions  given  below.  The  volume  of  fluid  present  in  the  stomach 
one  hour  after  an  Ewald  meal  varies  under  normal  conditions  between  50  and  100 
c.c.  In  cases  of  hypersecretion  or  defective  motility  200-300  c.c.  may  be  found. 
Very  excessive  volumes,  e.g.,  500-3000  c.c,  are  indicative  of  dilatation  of  the  stomach 
and  suggest  pyloric  stenosis,  either  benign  or  malignant. 

I.  Total  Acidity.'* — Add  3  drops  of  a  i  per  cent  alcoholic  solution  of  phenol- 
phthalein^  to  the  contents  of  vessel  A  and  titrate  with  N/io  sodium  hydroxide  solu- 
tion until  a  faint  pink  color  is  produced  and  persists  for  almost  two  minutes.  Take 
the  burette  reading  and  calculate  the  total  acidity. 

Calculation. — The  total  acidity  may  be  expressed  in  the  following  ways: 

I.  The  number  of  cubic  centimeters  of  N/io  sodium  hydroxide  solution  neces- 
sary to  neutralize  100  c.c.  of  gastric  juice. 

2;  The  weight  (in  grams)  of  sodium  hydroxide  necessary  to  neutralize  100  c.c. 
of  gastric  juice. 

3.  The  weight  (in  grams)  of  hydrochloric  acid  which  the  total  acidity  of  100 
c.c.  of  gastric  juice  represents,  i.e.,  percentage  of  hydrochloric  acid. 

The  forms  of  expression  most  frequently  employed  are  i  and  3,  preference  being 
given  to  the  former,  particularly  in  clinical  work. 

In  making  the  calculation  note  the  number  of  cubic  centimeters  of  N/io  sodium 
hydroxide  required  to  neutralize  10  c.c.  of  the  gastric  juice  and  multiply  it  by  10  to 
obtain  the  number  of  cubic  centimeters  necessary  to  neutralize  100  c.c.  of  the  fluid. 

^  Phosphotungstic  acid 0.3  c.c. 

Concentrated  hydrochloric  acid i .  o  c.c. 

Alcohol  95  per  cent 20.0  c.c. 

Distilled  water  sufficient  to  make 100. o  c.c. 

-  For  a  discussion  of  combined  acid  see  chapter  on  Gastric  Digestion. 
3  If  sufficient  gastric  juice  is  not  available  it  may  be  diluted  with  water  or  a  smaller 
amount,  e.g.,  5  c.c,  taken  for  each  determination. 

*  This  includes  free  and  combined  acid  and  acid  salts. 

^  One  gram  of  phenolphthalein  dissolved  in  100  cc.  of  95  per  cent  alcohol. 


GASTRIC    ANALYSIS  1 75 

If  it  is  desired  to  express  the  acidity  of  100  cc.  of  gastric  juice  in  terms  of  hydro- 
chloric acid,  by  weight,  multiply  the  value  just  obtained  by  0.00365.^ 

2.  Free  Acidity  (Organic  and  Inorganic). — Add  3  drops  of  sodium  alizarin 
sulphonate  solution-  to  the  contents  of  vessel  B  and  titrate  with  X/io  sodium  hy- 
droxide solution  until  a  violet  color  is  produced.  In  this  titration  the  red  color, 
which  appears  after  the  tinge  of  yellow  due  to  the  addition  of  the  indicator  has 
disappeared,  must  be  entirely  replaced  by  a  distinct  violet  color.  Take  the  burette 
reading  and  calculate  the  free  acidity  due  to  organic  and  inorganic  acids. 

Calculation. — Since  the  indicator  used  reacts  to  both  organic  and  inorganic 
acids,  the  number  of  cubic  centimeters  of  N/io  sodium  hydroxide  used  indicates 
the  free  acidity  of  10  cc.  of  gastric  juice.  The  data  for  100  cc.  of  gastric  juice  may 
be  calculated  according  to  the  directions  given  under  Total  Acidity,  page  174. 

3.  Free  Hydrochloric  Acid.^ — Add  4  drops  of  di-methyl-amino-azobenzene 
(Topfer's  reagent)  solution'*  to  the  contents  of  the  vessel  C  and  titrate  with  X/io 
sodium  hydroxide  solution  until  the  initial  red  color  is  replaced  by  orange  yellow.^ 
Take  the  burette  reading  and  calculate  the  free  acidity. 

Calculation.— T\it  indicator  used  reacts  only  to  free  hydrochloric  acid,  hence  the 
number  of  cubic  centimeters  of  N/io  sodium  hydroxide  used  indicates  the  volume 
necessary  to  neutralize  the  free  hydrochloric  acid  of  10  cc.  of  gastric  juice.  To 
determine  the  data  for  100  cc  of  gastric  juice  proceed  according  to  the  directions 
given  under  Total  Acidity,  page  174. 

4.  Combined  Acidity. — This  value  may  be  obtained  by  subtracting  the  number 
of  cubic  centimeters  of  N/io  sodium  hydroxide  used  in  neutralizing  the  contents  of 
vessel  B  from  the  number  of  cubic  centimeters  of  N/io  sodium  hydroxide  used  in 
neutralizing  A.  The  data  for  100  cc  of  gastric  juice  may  be  calculated  according 
to  directions  given  under  Total  Acidity,  page  174. 

5.  Acidity  Due  to  Organic  Acids  and  Acid  Salts. — This  value  may  be  conven- 
iently calculated  by  subtracting  the  number  of  cubic  centimeters  of  N/io  sodium 
hydroxide  used  in  neutralizing  the  contents  of  vessel  C  from  the  number  of  cubic 
centimeters  of  N/io  sodium  hydroxide  solution  used  in  neutralizing  the  contents  of 
vessel  B.  The  remainder  indicates  the  number  of  cubic  centimeters  of  N/io 
sodium  hydroxide  solution  necessary  to  neutralize  the  acidity  due  to  organic  acids 
and  acid  salts  present  in  10  cc.  of  gastric  juice.  The  data  for  100  cc  of  gastric 
juice  may  be  calculated  according  to  directions  given  under  Total  Acidity,  page  174. 

^  One  CO.  of  N/io  hydrochloric  acid  contains  0.00365  gram  of  hydrochloric  acid. 
^  One  gram  of  sodium  alizarin  sulphonate  dissolved  in  100  cc.  of  water. 
^  Hydrochloric  acid  not  combined  with  protein  material. 
*  One-half  gram  dissolved  in  100  cc.  of  95  per  cent  alcohol. 

^  If  the  orange  yellow  color  appears  as  soon  as  the  indicator  is  added  it  denotes  the  ab- 
sence of  free  acid. 


CHAPTER  IX 

FATS 

Fats  occur  very  widely  distributed  in  the  plant  and  animal  king- 
doms, and  constitute  the  third  general  class  of  food-stuffs.  In  plant 
organisms  they  are  to  be  found  in  the  seeds,  roots,  and  fruit  while  each 
individual  tissue  and  organ  of  an  animal  organism  contains  more  or  less 
of  the  substance.  In  the  animal  organism  fats  are  especially  abundant 
in  the  bone  marrow  and  adipose  tissue.  They  contain  the  same  ele- 
ments as  the  carbohydrates,  i.e.,  carbon,  hydrogen,  and  oxygen,  but 
the  oxygen  is  present  in  smaller  percentage  than  in  the  carbohydrates 


Fig.  50. — Beef  Fat.     {Long.) 

and  the  hydrogen  and  oxygen  are  not  present  in  the  proportion  to  form 
water. 

Chemically  considered  the  fats  are  esters^  of  the  tri-atomic  alcohol, 
glycerol,  and  the  mono-basic  fatty  acids.  In  the  formation  of  these  fats 
three  molecules  of  water  result.  This  water  arises  by  the  replacement 
of  the  H's  of  the  carboxyl  groups  of  the  three  fatty  acid  molecules  by 
the  glycerol  radical,  thus  yielding  the  following  type  of  formula.  In 
this  case  the  combination  is  with  palmitic  acid  (C15H31COOH). 

CH2— OOCH31C15 
I 

CH-OOCH31C16. 
I 
CH2— OOCH31C15 

^  An  ester  is  an  oxyacid,  one  of  whose  acid  hydrogens  is  replaced  by  an  organic  radical. 

176 


FATS  177 

The  three  fatty  acid  radicals  entering  into  the  structure  of  a  neutral 
fat  may  be  the  radicals  of  the  same  fatty  acid  or  they  may  consist  of 
the  radicals  of  three  different  fatty  acids. 

By  hydrolysis  of  a  neutral  fat,  i.e.,  by  the  addition  to  the  molecule 
of  those  elements  which  are  eliminated  in  the  formation  of  the  fat  from 
glycerol  and  fatty  acid,  it  may  be  resolved  into  its  component  parts, 
i.e.,  glycerol  and  fatty  acid.  In  the  case  of  palmitin  the  following 
would  be  the  reaction: 

C3H5(OCi5H3iCO)3+3H20-^C3H5(OH)3+3(Ci5H3iCOOH). 

Palmitin.  Glycerol.  Palmitic  acid. 

This  process  is  called  saponification  and  may  be  produced  by  boiling 
with  alkalis;  by  the  action  of  steam  under  pressure;  by  long-continued 
contact  with  air  and  light;  by  the  action  of  certain  bacteria  and  by 
fat-splitting  enzymes  or  lipases,  e.g.,  pancreatic  lipase  (see  page  188). 
The  cells  forming  the  walls  of  the  intestines  evidently  possess  the  pecul- 
iar property  of  synthesizing  the  glycerol  and  fatty  acid  thus  formed  so 
that  after  absorption  these  bodies  appear  in  the  blood  not  in  their 
individual  form  but  as  neutral  fats. 

The  principal  animal  fats  wdth  \\±iich  we  have  to  deal  are  stearin, 
palmitin,  olein,  and  butyrin.  Such  less  important  forms  as  laurin  and 
myristin  may  occur  abundantly  in  plant  organisms.  The  older  system 
of  nomenclature  for  these  fats  was  to  apply  the  prefix  "tri"  in  each 
case  {e.g.,  /n-palmitin)  since  three  fatty  acid  radicals  are  contained  in 
the  neutral  fat  molecule. 

The  fatty  acids  corresponding  to  the  above-mentioned  animal  fats 
are  stearic,  CH3(CH2)i6COOH;  palmitic,  CH3(CH2)i4COOH;  oleic, 
CH3(CH2)7CH  =  CH(CH2)7COOH;  and  butyric,  CH3(CH2)2COOH. 
Stearic,  palmitic  and  butyric  acids  are  saturated  fatty  acids,  whereas 
oleic  acid  belongs  to  the  class  of  unsaturated  acids.  Linoleic  acid  is 
also  unsaturated.  Upon  the  presence  of  these  unsaturated  fatty  acids 
depends  the  property  which  certain  fats  possess  of  absorbing  or  combin- 
ing with  iodine.  The  determination  of  this  so-called  "iodine  absorption 
number"  is  important  in  the  differentiation  of  fats  and  oils.  Fats 
containing  the  unsaturated  acids  oleic  and  linoleic  may  be  transformed 
by  "hydrogenation"^  into  the  fats  containing  the  corresponding 
saturated  acid  (stearic) .     The  oleic  acid  is  changed  thus : 

C18H34O2  +  2H— ^CisHseOa- 

Oleic  acid.  Stearic  acid. 

Fats  occur  ordinarily  as  mixtures  of  several  individual  fats.     For 
example,  the  fat  found  in  animal  tissues  is  a  mixture  of  olein,  palmitin 
*  Addition  of  hydrogen  to  the  molecule,  producing  a  "hydrogenated  fat." 


lyS  PHYSIOLOGICAL   CHEMISTRY 

and  stearin,  the  percentage  of  any  one  of  these  fats  present  depending 
upon  the  particular  species  of  animal  from  whose  tissue  the  fat  was 
derived.  Thus  the  ordinary  mutton  fat  contains  more  stearin  and  less 
olein  than  the  pork  fat.  Human  fat  contains  from  67  per  cent  to  85  per 
cent  of  olein  and,  according  to  Benedict  and  Osterberg,  upon  analysis 
yields  76.08  per  cent  of  carbon  and  11.78  per  cent  of  hydrogen.  Butter 
consists  in  large  part  of  olein  and  palmitin.  Stearin,  butyrin,  caproin 
and  traces  of  other  fats  are  also  present. 

Pure  neutral  fats  are  odorless,  tasteless,  and  generally  colorless. 
They  are  insoluble  in  the  ordinary  protein  solvents  such  as  water,  salt 
solutions,  and  dilute  acids  and  alkalis,  but  are  very  readily  soluble  in 
ether,  benzene,  chloroform,  and  boiling  alcohol.  The  neutral  fats  are 
non-volatile  substances  possessing  a  neutral  reaction.  If  allowed  to 
remain  in  contact  with  the  air  for  a  sufficient  length  of  time  they  become 
yellow  in  color,  assume  an  acid  reaction  and  are  said  to  be  rancid.  The 
neutral  fats  may  be  crystallized,  some  of  them  with  great  facility.  The 
crystalline  forms  of  some  of  the  more  common  fats  are  reproduced  in 
Figs.  50,  51  and  52  on  pages  176,  179  and  181.  Each  individual  fat 
possesses  a  specific  melting-  or  boiling-point  (according  to  whether  the 
body  is  solid  or  fluid  in  character),  and  this  property  of  melting  or  boiling 
at  a  definite  temperature  may  be  used  as  a  means  of  differentiation  in 
the  same  way  as  the  coagulation  temperature  (see  page  105)  is  used  for 
the  differentiation  for  coagulable  proteins.  When  shaken  with  water, 
or  a  solution  of  albumin,  soap,  or  acacia,  the  liquid  fats  are  finely  divided 
and  assume  a  condition  known  as  an  emulsion.  The  emulsion  with 
water  is  transitory,  while  the  emulsions  with  soap,  acacia,  or  albumin 
are  permanent. 

The  fat  ingested  continues  essentially  unaltered  until  it  reaches  the 
intestine  where  it  is  acted  upon  by  pancreatic  lipase  (steapsin),  the  fat- 
splitting  enzyme  of  the  pancreatic  juice  (see  page  188),  and  glycerol 
and  fatty  acid  are  formed.  The  glycerol  is  absorbed  directly.  The 
fatty  acid  thus  formed  unites  with  the  alkalis  of  the  pancreatic  juice 
and  forms  soluble  soaps.  These  soaps  are  readily  absorbed.  That 
bile  is  of  assistance  in  the  absorption  of  fat  is  indicated  by  the 
increase  of  fat  in  the  feces  when  for  any  reason  bile  does  not  pass 
into  the  intestine.  Bloor^  claims  that  neither  petroleum  hydrocar- 
bons nor  nonsaponifiable  esters,  e.g.,  wool  fat  (lanolin),  are  absorbed. 
He  believes  that  saponification  is  a  necessary  preliminary  to  absorption. 

The  fat  distributed  throughout  the  animal  body  is  formed  partly 
from  the  ingested  fat  and  partly  from  carbohydrates  and  the  "carbon 

^  Bloor:  Jour.  Biol.  Chem.,  15,  105,  1913. 


FATS  179 

moiety"  of  protein  material.  The  formation  of  adipocere^  and  the 
occurrence  of  fatty  degeneration  are  sometimes  given  as  proofs  of  the 
formation  of  fat  from  protein.  This  is  questioned  by  many  investiga- 
tors. Rather  more  satisfactory  and  direct  proof  of  the  formation  of  fat 
from  protein  material  has  been  obtained  by  Hofmann  in  experimentation 
with  fly-maggots.  The  normal  content  of  fat  in  a  number  of  maggots 
was  determined  and  later  the  fat  content  of  others  which  had  developed 
in  blood  (84  per  cent  of  the  solid  matter  of  blood  plasma  is  protein 
material)  was  determined.  The  fat  content  was  found  to  have  in- 
creased 700  to  HOC  per  cent  as  a  result  of  the  diet  of  blood  proteins. 


Fig.  51. — Mutton  Fat.     (Long.) 

The  celebrated  experiments  of  Pettenkofer  and  Voit,  however,  have 
furnished  what  is,  perhaps,  the  most  substantial  positive  e\ddence  of 
the  formation  of  fat  from  protein.  These  investigators  fed  dogs  large 
amounts  of  lean  meat,  daily,  and  through  examination  of  urine,  feces  and 
expired  air  were  enabled  to  account  for  only  part  of  the  ingested  carbon, 
although  obtaining  a  satisfactory  nitrogen  balance.  The  discrepancy 
in  the  carbon  balance  was  explained  upon  the  theory  that  the  protein  of 
the  ingested  meat  had  been  split  into  a  nitrogenons  and  a  non-nitroge- 
nous portion  in  the  organism,  and  that  the  non-nitrogenous  portion,  the 
so-called  "carbon  moiety"  of  the  protein,  had  been  subsequently  trans- 
formed into  fat  and  deposited  as  such  in  the  tissues  of  the  organism. 
Later  evidence  in  favor  of  the  formation  of  fat  from  protein  has 
been  furnished  by  the  experiments  of  Weinland.  This  investigator 
worked  with  the  larvae  of  Calliphorar  these  larvae  being  rubbed  up 
in  a  mortar^  with  Witte's  peptone  and  water  to  form  a  homogeneous 

'  A  very  complete  analysis  of  adipocere  was  reported  by  Ruttan  and  Marshall  before 
the  Society  of  Biological  Chemists,  Boston,  Dec.  27,  1915. 
^  The  ordinary  "blow-fly." 


l8o  PHYSIOLOGICAL   CHEMISTRY 

mixture.  After  placing  these  mixtures  at  38°C.  for  24  hours  the  fat 
content  was  found  to  have  increased,  as  much  as  140  per  cent  in  some 
instances.  The  active  agency  in  this  transformation  of  fat  is  the  larval 
tissue,  since  the  tissues  of  both  the  dead  and  living  larvae  possess  the 
property.  Data  are  given  from  control  tests  which  show  that  the  action 
of  bacteria  in  this  transformation  of  protein  was  excluded. 

Some  investigators  are  not  inclined  to  accept  any  data  regarding  the 
formation  of  fat  from  protein  as  conclusive. 

Experiments  on  Fats 

1.  Solubility. — Test  the  solubility  of  olive  oil  in  water,  dilute  acid  and  alkali 
and  in  cold  alcohol,  hot  alcohol,  chloroform,  ether,  and  carbon  tetrachloride. 

2.  Formation  of  a  Transparent  Spot  on  Paper. — Place  a  drop  of  olive  oil 
upon  a  piece  of  ordinary  writing  paper.  Note  the  transparent  appearance  of  the 
paper  at  the  point  of  contact  with  the  fat. 

3.  Reaction. — ^Try  the  reaction  of  fresh  olive  oil  to  litmus,  Congo  red  and  phe- 
nolphthalein.  Repeat  the  test  with  rancid  olive  oil.^  What  is  the  reaction  of  a 
fresh  fat  and  how  does  this  reaction  change  upon  allowing  the  fat  to  stand  for  some 
time? 

4.  Formation  of  Acrolein. — To  a  Uttle  oUve  oil  in  a  mortar  add  some  dry  potas- 
sium bisulphate,  EIHSO4,  and  rub  up  thoroughly.  Transfer  to  a  dry  test-tube 
and  cautiously  heat.  Note  the  irritating  odor  of  acrolein.  The  glycerol  of  the 
fat  has  been  dehydrolyzed  and  acrylic  aldehyde  or  acrolein  has  been  produced. 
This  is  the  reaction  which  takes  place : 

CH2OH         CHO 

CHOH    -^  CH+2H2O. 

I  II 

CH2OH        CH2 

Glycerol.  Acrolein. 

5.  Emulsification. — (a)  Shake  up  a  drop  of  neutral'  oUve  oil  with  a  little  water 
in  a  test-tube.  The  fat  becomes  finely  divided,  forming  an  emulsion.  This 
is  not  a  permanent  emulsion  since  the  fat  separates  and  rises  to  the  top  upon 
standing. 

(b)  To  5  c.c.  of  water  in  a  test-tube  add  2  or  3  drops  of  0.5  per  cent  Na2C03. 
Introduce  into  this  faintly  alkaline  solution  a  drop  of  neutral  oUve  oil  and  shake. 
The  emulsion  while  not  permanent  is  not  so  transitory  as  in  the  caes  of  water  free 
from  sodium  carbonate. 

(c)  Repeat  (b)  using  rancid  olive  oil.    What  sort  of  an  emulsion  do  you  get 

'  Intact  larvae  were  used  in  some  experiments. 

*  To  prepare  rancid  olive  oil  add  5  drops  of  oleic  acid  to  10  c.c.  of  olive  oil. 

^  Neutral  olive  oil  may  be  prepared  by  shaking  ordinary  olive  oil  with  a  lo  per  cent 
solution  of  sodium  carbonate.  This  mixture  should  then  be  extracted  with  ether  and  the 
ether  removed  by  evaporation.     The  residue  is  neutral  olive  oil. 


FATS 


I»I 


and  why?    It  is  impossible  to  emulsify  a  highly  rancid  fat  due  to  the  excessive 
formation  of  rather  insoluble  soaps  about  the  oil  drops. 

(d)  Shake  a  drop  of  neutral  olive  oil  with  dilute  albumin  solution.  What  is 
the  nature  of  this  emulsion?    Examine  it  under  the  microscope. 

6.  Fat  Crystals. — Dissolve  a  small  piece  of  lard  in  ether  in  a  test-tube,  add 
an  equal  volimie  of  alcohol  and  allow  the  alcohol-ether  mixture  to  evaporate 
spontaneously.  Examine  the  crystals  under  the  microscope  and  compare  them 
with  those  reproduced  in  Figs.  50,  51,  and  52,  on  pages  176,  179  and  181. 

7.  Saponification  of  Bayberry  Tallow.' — Fill  a  large  casserole  two-thirds 
full  of  water  rendered  strongly  alkaUne  with  soUd  potassium  hydroxide  (a.  stick 
one  inch  in  length).  Add  about  10  grams  of  bayberry  tallow  and  boil,  keeping 
the  volume  constant  by  adding  water  as  needed.    When  saponification  is  com- 


PoRK  Fat. 


plete^  remove  25  c.c.  of  the  soap  solution  for  use  in  Experiment  8  and  add  concen- 
trated hydrochloric  acid  slowly  to  the  remainder  until  no  further  precipitate  is 
produced.'  Cool  the  solution  and  the  precipitate  of  free  fatty  acid  will  rise  to  the 
surface  and  form  a  cake.  In  this  instance  the  fatty  acid  is  principally  palmitic 
acid.  Remove  the  cake,  break  it  into  small  pieces,  wash  it  with  water  by  decan- 
tation  and  transfer  to  a  small  beaker  by  means  of  95  per  cent  alcohol.  Heat  on  a 
water-bath  imtil  the  palmitic  acid  is  dissolved,  then  filter  through  a  dry  filter 
paper  and  allow  the  filtrate  to  cool  slowly  in  order  to  obtain  satisfactory  crystals. 
Write  the  reactions  which  have  taken  place  in  this  experiment. 

When  the  palmitic  acid  has  completely  crystallized  filter  off  the  alcohol,  dry 
the  crystals  between  filter  papers  and  try  the  tests  given  in  Experiment  10,  p.  182. 

8.  Salting-out  Experiments. — To  25  c.c.  of  soap  solution,  prepared  as  de- 
scribed above,  add  soUd  sodiimi  chloride  to  the  point  of  saturation,  with  continual 
stirring.    A  menstruum  is  thus  formed  in  which  the  soap  is  insoluble.     This 

1  Bayberry  tallow  is  derived  from  the  fatty  covering  of  the  berries  of  the  u-ax  myrtle.  It 
is  therefore  frequently  called  "myrtle  wax"  or  "bayberry  wax." 

^  Place  2  or  3  drops  in  a  test-tube  full  of  water.  If  saponification  is  complete  the  prod- 
ucts will  remain  in  solution  and  no  oil  will  separate. 

'Under  some  conditions  a  purer  product  is  obtained  if  the  soap  solution  is  cooled  before 
precipitating  the  fatty  acid. 


I»2 


PHYSIOLOGICAL    CHEMISTRY 


salting-out  process  is  entirely  analogous  to  the  salting-out  of  proteins  (see  page 
102). 

9.  Formation  of  Insoluble  Soaps. — Introduce  5  c.c.  of  soap  solution  into  each 
of  two  test-tubes.  To  the  contents  of  one  tube  add  a  small  amount  of  a  solution 
of  calcium  chloride  and  to  the  contents  of  the  other  tube  add  a  small  amount 
of  a  solution  of  magnesimn  sulphate.  Note  the  formation  of  insoluble  soaps  of 
calcimn  and  magnesium. 

10.  Palmitic  Acid. — (a)  Examine  the  crystals  under  the  microscope  and  com- 
pare them  with  those  shown  in  Fig.  53,  below. 

(b)  SolubiUty. — ^Try  the  solubiUty  of  palmitic  acid  in  the  same  solvents  as  used 
on  fats  (see  page  180). 

(c)  Melting-point. — Determine  the  melting-point  of  palmitic  acid  by  one  of 
the  methods  given  on  page  183. 


Fig.  53.^ — Palmitic  Acid. 

(d)  Formation  of  Transparent  Spot  on  Paper. — Melt  a  httle  of  the  fatty  acid 
and  allow  a  drop  to  fall  upon  a  piece  of  ordinary  writing  paper.  How  does  this 
compare  with  the  action  of  a  fat  under  similar  circumstances? 

(e)  Acrolein  Test. — ^Apply  the  test  as  given  under  4,  page  180.  Explain  the 
result. 

(f)  Iodine  Absorption  Test. — For  directions  see  Experiment  13. 

11.  Saponification  of  Lard. — To  25  grams  of  lard  in  a  fiask  add  75  c.c.  of 
alcohoUc -potash  solution  and  warm  upon  a  water-bath  until  saponification  is 
complete.  (This  point  is  indicated  by  the  complete  solubility  of  a  drop  of  the 
solution  when  allowed  to  fall  into  a  Uttle  water.)  Now  transfer  the  solution  from 
the  flask  to  an  evaporating  dish  containing  about  100  c.c.  of  water  and  heat  on  a 
water-bath  until  all  the  alcohol  has  been  driven  off.  Precipitate  the  fatty  acid 
with  hydrochloric  acid  and  cool  the  solution.  Remove  the  fatty  acid  which  rises 
to  the  surface,^  neutralize  the  solution  with  sodiimi  carbonate  and  evaporate  to 
dryness.  Extract  the  residue  with  alcohol,  remove  the  alcohol  by  evaporation 
upon  a  water-bath  and  on  the  residue  of  glycerol  thus  obtained  make  the  tests 
as  given  below. 

12.  Glycerol,     (a)  Taste.— What  is  the  taste  of  glycerol? 

^  After  drying  the  acid  make  an  iodine  absorption  test  as  described  in  Experiment  13. 


FATS 


183 


Oe 


(b)  Solubility. — Try  the  solubility  of  glycerol  in  water,  alcohol  and  ether. 

(c)  H5rpochlorite-Orcinol  Reaction.' — This  is  based  on  the  oxida- 
tion of  glycerol  to  the  corresponding  aldose  sugar  glycerose  and  the 
detection  of  the  latter  by  means  of  orcinol.  Homologues  of  glycerol 
as  well  as  the  corresponding  acids  and  certain  sugars  as  glucose  and 
mannose  give  the  reaction.  The  first  named  occur  seldom  while  the 
latter  may  be  removed  with  baryta. 

Two  to  3  c.c.  of  a  I  per  cent  or  Ko  per  cent  solution  of  glycerol  in  water  is 
treated  with  exactly  3  drops  ( =  0.12  c.c.)  normal  NaOCl-  and  boiled  for  a  minute. 
To  the  Uquid  while  still  hot  add  3  drops  of  hydrochloric  acid  (sp.  gr.  1.124)  and 
boil  30-60  seconds  to  drive  off  chlorine,  a 
colorless  solution  being  obtained.  Then  add 
an  equal  volume  of  f xmiing  hydrochloric  acid  and 
a  small  knife -point  of  orcinol.  On  boiling  the 
mixture  becomes  a  beautiful  violet  or  green  blue. 
The  precipitate  formed  is  soluble  in  amyl  alcohol 
and  may  be  examined  spectroscopically. 

(d)  Acrolein  Test. — Repeat  the  test  as  given 
under  4,  page  180. 

(e)  Borax  Fusion  Test. — Fuse  a  little 
glycerol  on  a  platinum  wire  with  some  powdered 
borax  and  note  the  characteristic  green  flame. 
This  color  is  due  to  the  glycerol  ester  of  boric 
acid. 

(f)  Fehling's  Test. — ^How  does  this  result 
compare  with  the  results  on  the  sugars? 

(g)  Solution  of  Cu(OH)2.  Form  a  little 
cupric  hydroxide  by  mixing  copper  sulphate 
and  potassium  hydroxide.  Add  a  little  glycerol 
to  this  suspended  precipitate  and  note  what 
occurs. 

13.  Iodine  Absorption  Test. — Dissolve  a 
small  amount  of  an  unsaturated  organic  acid, 
e.g.,  oleic  acid,  in  chloroform.  Add  2-3  drops 
of  Hiibl's  iodine  solution^  and  shake.  The  solu- 
tion will  be  decolorized  if  unsatiirated  acids  are 
present.  This  is  due  to  the  absorption  of  the 
iodine.  The  test  should  be  controlled  by  shak- 
ing chloroform  and  iodine  solution  to  which  no 
acid  has  been  added. 

14.  Melting-point  of  Fat.— First  Method. — 

Insert  one  of  the  melting-point  tubes,  furnished  by  the  instructor,  into  the 
liquid  fat  and  draw  up  the  fat  until  the  bulb  of  the  tube  is  about  one -half  full 
of  the  material.     Then  fuse  one  end  of  the  tube  in  the  flame  of  a  Bunsen  burner 

'  Mandel  and  Neuberg:  Block.  Zcil.,  71,  214,  1915. 
^  Made  according  to  Raschig:  Bcr.,  40,  4586,  1907. 

'  Prepared  by  dissolving  26  grams  of  iodine  and  30  grams  of  mercuric  chloride  in  one 
liter  of  95  per  cent  alcohol. 


OS 


Fig.  54. — Melting-point 
Apparatus. 


184  PHYSIOLOGICAL    CHEMISTRY 

and  fasten  the  tube  to  a  thermometer  by  means  of  a  rubber  band  in  such  a  maimer 
that  the  bottom  of  the  fat  col'omn  is  on  a  level  with  the  bulb  of  the  thermometer 
(Fig.  54,  p.  183).  Fill  a  beaker  of  medium  size  about  two-thirds  full  of  water  and 
place  it  within  a  second  larger  beaker  which  also  contains  water,  the  two  vessels 
being  separated  by  pieces  of  cork.  Immerse  the  bulb  of  the  thermometer  and 
the  attached  tube  in  such  a  way  that  the  bulb  is  about  midway  between  the 
upper  and  the  lower  surfaces  of  the  water  of  the  inner  beaker.  The  upper 
end  of  the  tube  being  open  it  must  extend  above  the  surface  of  the  surround- 
ing water.  Apply  gentle  heat,  stir  the  water,  and  note  the  temperature  at 
which  the  fat  first  begins  to  melt.  This  point  is  indicated  by  the  initial  trans- 
parency. For  ordinary  fats,  raise  the  temperature  very  cautiously  from  30°C. 
To  determine  the  congealing-point  remove  the  flame  and  note  the  temperature 
at  which  the  fat  begins  to  soUdify.  Record  the  melting-  and  congealing-points 
of  the  various  fats  submitted  by  the  instructor. 

Second  Method. — Fill  a  small  evaporating  dish  about  one-half  full  of  mercury 
and  place  it  on  a  water-bath.  Put  a  small  drop  of  the  fat  under  examination  on  an 
ordinary  cover-glass  and  place  this  upon  the  surface  of  the  mercury.  Raise  the 
temperature  of  the  water-bath  slowly  and  by  means  of  a  thermometer  whose  bulb 
is  immersed  in  the  mercury,  note  the  melting-point  of  the  fat.  Determine  the  con- 
gealing-point by  removing  the  flame  and  leaving  the  fat  drop  and  cover-glass  in 
position  upon  the  mercury.  How  do  the  melting-points  as  determined  by  this 
method  compare  with  those  as  determined  by  the  first  method?  Which  method 
is  the  more  accurate,  and  why? 


CHAPTER  X 
'      PANCREATIC  DIGESTION^ 

As  soon  as  the  food  mixture  leaves  the  stomach  it  comes  into  inti- 
mate contact  with  the  bile  and  the  pancreatic  juice.  Since  these  fluids 
are  alkaline  in  reaction  (see  Bile,  page  202)  there  can  obviously  be  no 
further  peptic  activity  after  they  have  become  intimately  mixed  with 
the  chyme  and  have  neutralized  the  acidity  previously  imparted  to  it 
by  the  hydrochloric  acid  of  the  gastric  juice.  The  pancreatic  juice 
reaches  the  intestine  through  the  duct  of  Wirsung  which  opens  into  the 
intestine  near  the  pylorus. 

Normally  the  secretion  of  pancreatic  juice  is  brought  about  by  the 
stimulation  produced  by  the  acid  chyme  as  it  enters  the  duodenum. 
Therefore,  any  factor  which  produces  an  increased  flow  of  gastric  juice 
such,  for  example,  as  water^  will  cause  a  stimulation  of  the  pancreatic 
secretion.  The  secretion  of  pancreatic  juice  is  probably  not  due  to  a 
nervous  reflex  as  was  believed  by  Pawlow  but  rather,  as  Bayliss  and 
Starling  have  shown,  is  dependent  upon  the  presence,  in  the  epithelial 
cells  of  the  duodenum  and  jejunum  of  a  body  known  as  prosecretin. 
This  body  is  changed  into  secretin  through  the  hydrolytic  action  of  the 
acid  present  in  the  chyme.  The  secretin  is  then  absorbed  by  the  blood, 
passes  to  the  pancreas  and  stimulates  the  pancreatic  cells,  causing  a 
flow  of  pancreatic  juice.  The  quantity  of  juice  secreted  under  these 
conditions  is  proportional  to  the  amount  of  secretin  present.  The 
activity  of  secretin  solutions  is  not  diminished  by  boiling,  hence  the 
body  does  not  react  like  an  enzyme.  Further  study  of  the  body  may 
show  it  to  be  a  definite  chemical  individual  of  relatively  low  molecular 
weight.  It  has  not  been  possible  thus  far  to  obtain  secretin  from  any 
tissues  except  the  mucous  membrane  of  the  duodenum  and  jejunum. 

This  secretin  mentioned  above  belongs  to  the  class  of  substances 
called  hormones  or  chemical  messengers.  These  hormones  play  a  very 
important  part  in  the  coordination  of  the  activities  of  certain  functions 
and  glands.  Other  important  hormones  are  those  elaborated  by  the 
thyroids,  the  adrenals,  the  pituitary  body  (hypophysis),  the  embryo  and 

^  Under  this  head  we  will  consider  only  such  digestive  processes  as  are  brought  about 
by  enzymes  originating  in  the  pancreas.  In  the  following  chapter  on  Intestinal  Digestion 
will  be  found  a  consideration  of  such  enzymes  as  have  a  true  intestinal  origin. 

*  See  chapter  on  Gastric  Digestion. 

185 


1 86  PHYSIOLOGICAL    CHEMISTRY 

the  reproductive  glands.  It  is  claimed  by  some  that  all  active  organs 
of  the  body  produce  hormones. 

The  juice  as  obtained  from  a  permanent  fistula  differs  greatly  in 
its  properties  from  the  juice  as  obtained  from  a  temporary  fistula,  and 
neither  form  of  fluid  possesses  the  properties  of  the  normal  fluid.  Pan- 
creatic juice  collected  by  Glaessner  from  a  natural  fistula  has  been 
found  to  be  a  colorless,  clear,  strongly  alkaline  fluid  which  foams  readily. 
It  is  "further  characterized  by  containing  albumin,  globulin,  proteose, 
and  peptone;  nucleoprotein  is  also  present  in  traces.^  The  average 
daily  secretion  of  pancreatic  juice  is  650  c.c.  and  its  specific  gravity  is 
1.008.  The  fluid  .contains  1.3  per  cent  of  solid  matter  and  the  freezing- 
point  is  —  o.47°C.  The  normal  pancreatic  secretion  contains  at  least 
four  distinct  enzymes.  They  are  trypsin,  a  proteolytic  enzyme;  pan- 
creatic amylase  (amylopsin),  an  amylolytic  enzyme;  pancreatic  lipase 
(steapsin) ,  a  fat-splitting  enzyme ;  and  pancreatic  rennin,  a  milk-coagu- 
lating enzyme. 

The  most  important  of  the  four  enzymes  of  the  pancreatic  juice  is 
the  proteolytic  enzyme  trypsin.  This  enzyme  resembles  pepsin  in  so 
far  as  each  has  the  power  of  breaking  down  protein  material,  but  the 
trypsin  has  much  greater  digestive  power  and  is  able  to  cause  a  more 
complete  decomposition  of  the  complex  protein  molecule.  In  the 
process  of  normal  digestion  the  protein  constituents  of  the  diet  are  for 
the  most  part  transformed  into  proteoses  (albumoses)  and  peptones 
before  coming  in  contact  with  the  enzyme  trypsin.  This  is  not  abso- 
lutely essential,  however,  since  trypsin  possesses  digestive  activity  suffi- 
cient to  transform  unaltered  native  proteins  and  to  produce  from  their 
complex  molecules  comparatively  simple  fragments.  Among  the  prod- 
ucts of  tryptic  digestion  are  ^ro/eo5ej,  peptones,  peptides,  leucine,  tyrosine, 
aspartic  acid,  glutamic  acid,  alanine,  phenylalanine,  glycocoll,  cystine, 
serine,  valine,  proline,  oxyproline,  isoleucine,  arginine,  lysine,  histidine, 
and  tryptophane.  (The  crystalline  forms  of  many  of  these  products  are 
reproduced  in  Chapter  IV.)  Trypsin  does  not  occur  preformed  h^  the 
gland,  but  exists  there  as  a.  zymogen  called  trypsinogen  which  bears  the 
same  relation  to  trypsin  that  pepsinogen  does  to  pepsin.  Trypsin  has 
never  been  obtained  in  a  pure  form  and  therefore  very  little  can  be 
stated  definitely  as  to  its  nature.  The  enzyme  is  the  most  active  in 
alkaline  solution  but  is  also  active  in  neutral  or  slightly  acid  solutions. 
Trypsin  is  destroyed  by  mineral  acids  and  may  also  be  destroyed  by 
comparatively  weak  alkali  (2  per  cent  sodium  carbonate)  if  left  in  con- 
tact for  a  sufficiently  long  time.  Trypsinogen,  on  the  other  hand,  is 
more  resistant  to  the  action  of  alkalis.     In  pancreatic  digestion  the  pro- 

^  Glaessner:  Zeitschrift  jUr  physiologische  Cheniie,  40,  476,  1904. 


PANCREATIC   DIGESTION  1 87 

tein  does  not  swell  as  is  the  case  in  gastric  digestion,  but  becomes  more 
or  less  "honey-combed"  and  finally  disintegrates. 

The  presence  of  active  pepsin  in  the  contents  of  the  intestine  has 
been  demonstrated  by  Abderhalden  and  Meyer. ^  It  may  possibly  be 
that  pepsin  may  play  a  part  in  the  profound  intestinal  proteolysis  which 
has  up  to  this  time  been  assigned  to  trypsin  and  erepsin  (see  chapter  on 
Gastric  Digestion). 

The  pancreatic  juice  which  is  collected  by  means  of  a  fistula  pos- 
sesses- practically  no  power  to  digest  protein  matter.  A  body  called 
enter okinase  occurs  in  the  intestinal  juice  and  has  the  power  of  converting 
trypsinogen  into  trypsin.  This  process  is  known  as  the  "  activation"  of 
trypsinogen  and  through  it  a  juice  which  is  incapable  of  digesting  pro- 
tein may  be  made  active.  (For  further  discussion  of  enterokinase 
see  chapter  on  Intestinal  Digestion.)  Mendel  and  Rettger-  and  others 
have  demonstrated  that  activation  of  tr^-psinogen  into  trypsin  may  be 
brought  about  in  the  gland  as  well  as  in  the  intestine  of  the  li\'ing 
organism.  The  manner  of  the  activation  in  the  gland  and  the  nature 
of  the  body  causing  it  are  unknown  at  present.  Prym'^  denies  that 
such  an  activation  occurs. 

Delezenne  claims  that  trypsinogen  may  be  activated  by  soluble 
calcium  salts.  He  reports  experiments  which  indicate  that  proteolytic- 
ally  inactive  pancreatic  juice,  obtained  directly  from  the  duct,  when 
treated  \vith  salts  of  this  character,  assumes  the  property  of  digesting 
protein  material.  This  process  by  which  the  tripsinogen  is  activated 
through  the  instrumentality  of  calcium  salts  is  very  rapid  and  is  desig- 
nated by  Delezenne  as  an  "explosion."  The  suggestion  of  Mays  that 
there  may  possibly  be  several  precursors  of  tripsin  otie  of  which  is 
activated  by  enterokinase  and  the  others  by  other  agents,  is  of  interest 
in  this  connection. 

Boldyreff ^  has  demonstrated  the  presence  of  trypsin  in  the  stomach 
due  to  the  regurgitation  of  duodenal  contents  through  the  pylorus 
(see  Chapters  VII  and  VIII).  Others^  have  confirmed  this  finding  (see 
chapter  on  Gastric  Analysis). 

Pancreatic  amylase  {amylopsin),  the  second  of  the  pancreatic  en- 
zymes, is  an  amylolytic  enzyme  which  possesses  somewhat  greater  diges- 
tive power  than  the  salivary  amylase  (ptyalin)  of  the  saliva.  As  its 
name  implies,  its  acti\Tty  is  confined  to  the  starches,  and  the  products 
of  its  amylolytic  action  are  dextrins  and  sugar.     The  sugar  is  principally 

^Abderhalden  and  Meyer:  Zeit.  physiol.  C/tem.,  74,  67,  191 1. 
-Mendel  and  Rettger:  American  Journal  of  Physiology,  7. 
'  Prym:  Pfliiger^s  Archiv,  104  and  107. 

<Boldyreff:  Transactions  of  the  nth  Pirogoff's  Congress  of  Physicians,  St.  Petersburg, 
1910. 

'Spencer,  Meyer,  Rehfuss  and  Hawk:  American  Jour.  Physiol.,  39,  459,  1916. 


1 88  PHYSIOLOGICAL   CHEMISTRY 

maltose  and  this  by  the  further  action  of  an  inverting  enzyme  (maltase) 
is  transformed  into  glucose. 

It  is  possible  that  the  saliva  as  a  digestive  fluid  is  not  absolutely 
essential.  The  salivary  amylase  (ptyalin)  is  destroyed  by  the  hydro- 
chloric acid  of  the  gastric  juice  and  is  therefore  inactive  when  the  chyme 
reaches  the  intestine.  Should  undigested  starch  be  present  at  this 
point,  however,  it  would  be  quickly  transformed  by  the  active  pancreatic 
amylase.  This  enzyme  is  not  present  in  the  pancreatic  juice  of  infants 
during  the  first  few  weeks  of  life,  thus  showing  very  clearly  that  a  starchy 
diet  is  not  normal  for  this  period. 

The  pronounced  influence  of  electrolytes  upon  the  action  of  pancrea- 
tic amylase  and  other  amylases  has  been  demonstrated  many  times. ^ 
In  fact  the  removal  of  electrolytes  from  pancreatic  juice  by  dialysis 
yields  a  juice  which  possesses  no  power  to  split  starch.  It  also  appears 
that  the  CI  or  Br  ion  is  absolutely  essential  to  the  activity  of  animal 
amylases.^ 

It  has  been  claimed  that  pancreatic  amylase  has  a  slight  digestive 
action  upon  unboiled  starch. 

The  extent  to  which  amylase  is  present  in  the  feces  has  been  taken  as 
the  index  of  pancreatic  activity. 

The  third  enzyme  of  the  pancreatic  juice  is  called  pancreatic  lipase 
{steapsin)  and  is  a  fat-splitting  enzyme.  It  has  the  power  of  splitting 
the  neutral  fats  of  the  food  by  hydrolysis,  into  fatty  acid  and  glycerol. 
A  typical  reaction  would  be  as  follows: 

C3H5(OCi5H3iCO)3+3H20^3(Ci5H3iCOOH)+C3H5(OH)3. 

Palmitin.  Palmitic  acid.  Glycerol. 

Recent  researches  make  it  probable  that  fats  undergo  saponifica- 
tion to  a  certain  extent  prior  to  their  absorption.  The  fatty  acids 
formed  unite  with  the  alkalis  of  the  pancreatic  juice  and  intestinal 
secretion  to  form  soluble  soaps  which  are  readily  absorbed.  It  was 
formerly  believed  that  the  fats  could  also  be  absorbed  in  emulsion — 
a  condition  promoted  by  the  presence  of  the  soluble  soaps.  After  ab- 
sorption the  fatty  acids  are  resynthesized  to  form  neutral  fats  with 
glycerol. 

Bloor^  has  reported  experiments  which  "make  it  extremely  probable 
that  fats  can  be  absorbed  only  in  water-soluble  form  and  that  saponi- 
fication is  a  necessary  preliminary  to  absorption."  Petroleum  hydro- 
carbons and  non-saponifiable  esters,  e.g.,  wool  fat  (lanolin)  were  un- 

^  For  the  literature  see  Kendall  and  Sherman:  Jour.  Am.  Cheni.  Soc,  32,  1087,  1910. 
*  Wohlgemuth:  Biochem.  Zeit.,  9,  10,  1908;  and  Kendall  and  Sherman:  Jour.  Am.  Chem. 
Soc,  32,  1087    1910.     Bierry:  Biochem.  Zeit.,  40,  357,  1912. 
'  Bloor:  Jour.  Biol.  Chem.,  15,  105,  1913. 


PANCREATIC   DIGESTION  1 89 

absorbed.  Bloor  further  claims^  that  in  the  absorption  of  fats  there  is 
a  tendency  toward  the  formation  of  a  uniform  chyle  fat,  presumably  the 
characteristic  body  fat  of  the  animal. 

Pancreatic  lipase  is  very  unstable  and  is  easily  rendered  inert  by  the 
action  of  acid.  For  this  reason  it  is  not  possible  to  prepare  an  extract 
having  a  satisfactory  fat-splitting  power  from  a  pancreas  which  has 
been  removed  from  the  organism  for  a  sufficiently  long  time  to  have 
become  acid  in  reaction. 

The  fourth  enzyme  of  the  pancreatic  juice  is  called  pancreatic  rennin. 
It  is  a  milk-coagulating  enzyme  whose  action  is  very  similar  to  that 
of  the  gastric  rennin  found  in  the  gastric  juice.  It  is  supposed  to  show 
its  greatest  activity  at  a  temperature  varying  from  60°  to  65°C. 

PREPARATION  OF  AN  ARTIFICIAL  PANCREATIC  JUICE^ 

After  removing  the  fat  from  the  pancreas  of  a  pig  or  sheep,  finely  divide  the 
organ  by  means  of  scissors  and  grind  it  in  a  mortar.  If  convenient,  the  use  of  an 
ordinary  meat  chopper  is  a  very  satisfactory  means  of  preparing  the  pancreas. 

When  finely  divided  as  above  the  pancreas  should  be  placed  in  a  500  c.c. 
flask,  about  150  c.c.  of  30  per  cent  alcohol  added  and  the  flask  and  contents  shaken 
frequently  for  24  hours.  (What  is  the  reaction  of  this  alcoholic  extract  at  the  end 
of  this  period,  and  why?)  Strain  the  alcohoUc  extract  through  cheese  cloth, 
filter,  nearly  neutraUze  with  potassiimi  hydroxide  solution  and  then  exactly 
neutrahze  it  with  0.5  per  cent  sodium  carbonate. 

Products  of  Tryptic  Digestion 

Take  about  200  grams  of  lean  beef  which  has  been  freed  from  fat  and  finely 
ground  and  place  it  in  a  large-sized  beaker.  Introduce  equal  volumes  of  the  pan- 
creatic extract  prepared  as  above  and  0.5  per  cent  sodium  carbonate,  add  5  c.c. 
of  an  alcohoUc  solution  of  thymol  to  prevent  putrefaction,  and  place  the  beaker  in 
an  incubator  at  40°C.  Stir  the  contents  of  the  beaker  frequently  and  add  more 
thymol  if  it  becomes  necessary.  Allow  digestion  to  proceed  for  from  two  to  five 
days  and  then  separate  the  products  formed  as  follows:  Strain  off  the  undis- 
solved residue  through  cheese  cloth,  nearly  neutralize  the  solution  with  dilute 
hydrochloric  acid  and  then  exactly  neutraUze  it  with  0.2  per  cent  hydrochloric  acid. 
A  precipitate  at  this  point  would  indicate  alkaU  metaprotein  (alkali  albiuninate). 
Filter  off  any  precipitate  and  divide  the  filtrate  into  two  parts,  a  one-fourth  and  a 
three-fourth  portion. 

Transfer  the  one-fourth  portion  to  an  evaporating  dish  and  make  the  sepa- 
ration of  proteoses  and  peptones  as  weU  as  the  final  tests  upon  these  bodies 
according  to  the  directions  given  on  page  120. 

Place  about  5  c.c.  of  the  three -fourth  portion  in  a  test-tube  and  add  about 
I  c.c.  of  bromine  water.     A  violet  coloration^  indicates  the  presence  of  trypto- 

'  Bloor:  Jonr.  Biol.  Cliem.,  16,  517,  1914. 

-  For  other  methods  of  preparation  see  Karl  ^lays:  Zeitschrift  fur  physiologische  Chetnie, 
38,  428,  1903. 

*Kurajefif:  Zeit.  physiol.  Chetn.,  36,.  501,  1898-99. 


190  PHYSIOLOGICAL    CHEMISTRY 

phane  (see  page  77.  Also  see  glycyl-tryptophane  reaction  in  chapter  on  Intes- 
tinal Digestion).  To  another  5  c.c.  add  10  drops  of  concentrated  sulphuric  acid 
and  10  c.c.  of  a  10  per  cent  solution  of  mercuric  sulphate  in  5  per  cent  sulphuric 
acid.  Shake  the  tube  and  allow  to  stand  five  minutes.  A  yellow  precipitate  of  a 
mercury  compound  of  trjrptophane  forms. ^  Concentrate-  the  remainder  of  the 
three-fourths  portion  to  a  thin  syrup  and  make  the  separation  of  leucine  and  tyro- 
sine according  to  the  directions  given  on  page  85. 


GENERAL  EXPERIMENTS  ON  PANCREATIC  DIGESTION 

Experiments  on  Trypsin^ 

1.  The  Most  Favorable  Reaction  for  Tryptic  Digestion. — Prepare  seven  tubes 
as  follows : 

(a)  2-3  c.c.  of  neutral  pancreatic  extract +2-3  c.c.  of  water. 

(b)  2-3  c.c.  of  neutral  pancreatic  extract+2-3  c.c.  of  i  per  cent  sodium  car- 
bonate. 

(c)  2-3  c.c.  of  neutral  pancreatic  extract+2-3  c.c.  of  0.5  per  cent  sodium 
carbonate. 

(d)  2-3  c.c.  of  neutral  pancreatic  extract+2-3  c.c.  of  0.2  per  cent  hydro- 
chloric acid. 

(e)  2-3  c.c.  of  neutral  pancreatic  extract+2-3  c.c.  of  0.2  per  cent  combined 
hydrochloric   acid. 

(f)  2-3  c.c.  of  neutral  pancreatic  extract+2-3  c.c.  of  0.4  per  cent  boric  acid. 

(g)  2-3  c.c.  of  neutral  pancreatic  extract+2-3  c.c.  of  0.4  per  cent  acetic  acid. 
Add  a  small  piece  of  fibrin^  to  the  contents  of  each  tube  and  keep  them  at  40°C. 

noting  the  progress  of  digestion.  In  which  tube  do  we  find  the  most  satisfactory 
digestion,  and  why?  How  do  the  indications  of  the  digestion  of  fibrin  by  trypsin 
differ  from  the  indications  of  the  digestion  of  fibrin  by  pepsin? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the  following  series  of 
experiments  under  tryptic  digestion  use  the  neutral  extract  plus  an  equal  volume 
of  0.5  per  cent  sodium  carbonate.)  In  each  of  four  tubes  place  5  c.c.  of  alkaline 
pancreatic  extract.  Immerse  one  tube  in  cold  water  from  the  faucet,  keep  a 
second  at  room  temperature  and  place  a  third  in  the  incubator  or  water-bath  at 
40°C.  Boil  the  contents  of  the  fourth  for  a  few  moments,  then  cool  and  also  keep 
it  at  40°C.  Into  each  tube  introduce  a  small  piece  of  fibrin  and  note  the  progress 
of  digestion.  In  which  tube  does  the  most  rapid  digestion  occur?  What  is  the 
reason? 

3.  Influence  of  Bile. — Prepare  three  tubes  as  follows : 

(a)  5  c.c.  of  pancreatic  extract+i/2-1  c.c.  of  bile. 

(b)  5  c.c.  of  pancreatic  extract+5  c.c.  of  bile. 

'  It  has  been  claimed  that  a  similar  yellow  precipitate  forms  in  the  presence  of  tyrosine, 
cystine  and  polypeptides.  For  quantitative  estimation  of  tryptophane  see  Homer:  Jour. 
Biol.  Chetn.,  22,  369,  1915. 

2  If  the  solution  is  alkaline  in  reaction,  while  it  is  being  concentrated,  the  amino  acids 
will  be  broken  down  and  ammonia  will  be  liberated. 

'  For  these  experiments  as  well  as  for  those  on  the  other  pancreatic  enzymes  commer- 
cial preparations  of  trypsin  and  pancreatin  may  be  employed. 

^  Congo  red  fibrin  may  be  used  in  this  and  the  following  tests  on  tryptic  digestion.  If 
used  the  experiments  should  be  made  at  room  temperature.  For  preparation  of  this 
fibrin  see  Chapter  I. 


PANCREATIC   DIGESTION  IQI 

(c)   5  c.c.  of  pancreatic  extract. 

Introduce  into  each  tube  a  small  piece  of  fibrin  and  keep  them  at  40'C. 
Shake  the  tubes  frequently  and  note  the  -progress  of  digestion.  Does  the  pres- 
ence of  bile  retard  tryptic  digestion?  How  do  these  results  agree  with  those 
obtained  under  gastric  digestion? 

4.  Quantitative  Determination  of  Tryptic  Activity.^ — Gross'  Method. — This 
method  is  based  upon  the  principle  that  faintly  alkahne  solutions  of  casein  are 
precipitated  upon  the  addition  of  dilute  ( 1  per  cent  j  acetic  acid  whereas  its  digestion 
products  are  not  so  precipitated.  The  method  follows :  Prepare  a  series  of  tubes 
each  containing  10  c.c.  of  a  o.i  per  cent  solution  of  pure,  fat-free  casein,-  which 
has  been  heated  to  a  temperature  of  4o''C.  Add  to  the  contents  of  the  series  of 
tubes  increasing  amounts  of  the  trypsin  solution  under  examination,-  and  place 
them  at  40^0.  for  fifteen  minutes.  At  the  end  of  this  time  remove  the  tubes  and 
acidify  the  contents  of  each  with  a  few  drops  of  dilute  (i  per  centj  acetic  acid. 
The  tubes  in  which  the  casein  is  completely  digested  will  remain  clear  when 
acidified,  while  those  tubes  which  contain  undigested  casein  will  become  more 
or  less  txirbid  under  these  conditions.  Select  the  first  tube  in  the  series  which 
exhibits  no  turbidity  upon  acidification,  thus  indicating  complete  digestion  of 
the  casein,  and  calculate  the  tryptic  activity  of  the  enzyme  solution  under  ex- 
amination. 

Calcxilation. — The  unit  of  tryptic  activity  is  an  expression  of  the  power  of  i 
c.c.  of  the  fluid  under  examination  exerted  for  a  period  of  fifteen  minutes  on  10 
c.c.  of  a  0.1  per  cent  casein  solution.  For  example,  if  0.5  c.c.  of  a  trypsin  solu- 
tion completely  digests  10  c.c.  of  a  o.i  per  cent  solution  of  casein  in  fifteen 
minutes  the  activity  of  that  solution  would  be  expressed  as  follows : 

Tryptic  activity  =  1 -^ 0.5  =  2. 

Such  a  trypsin  solution  would  be  said  to  possess  an  activity  of  2.  If  0.3  c.c 
of  the  trypsin  solution  had  been  required  the  solution  would  be  said  to  possess  an 
activity  of  3.3 ;  i.e.,  i  -T-0.3  =3.3. 

Experiments  on  Pancreatic  Amylase 

I.  The  Most  Favorable  Reaction. — ^Prepare  seven  tubes  as  follows: 

(a)  I  c.c.  of  neutral  pancreatic  extract +  1  c.c.  of  starch  paste -1-2  c.c.  of 
water. 

(b)  I  c.c.  of  neutral  pancreatic  extract+i  c.c.  of  starch  paste +  2  c.c.  of 
I  per  cent  sodiimi  carbonate. 

(c)  I  c.c.  of  neutral  pancreatic  extract +i  c.c.  of  starch  paste +  2  c.c.  of 
0.5  per  cent  sodium  carbonate. 

(d)  I  c.c.  of  neutral  pancreatic  extract+i  c.c.  of  starch  paste +2  c.c.  of 
0.2  per  cent  hydrochloric  acid. 

Shake  each  tube  thoroughly  and  place  them  in  the  incubator  or  water-bath 
at  40°C.  At  the  end  of  a  half -hour  divide  the  contents  of  each  tube  into  two  parts 
and  test  one  part  by  the  iodine  test  and  the  other  part  by  Fehhng's  test.     Where 

'  For  a  discussion  of  Spencer's  method  for  the  quantitative  determination  of  tr\-psin 
in  stomach  contents  see  chapter  on  Gastric  Anal^'sis. 

*  Made  by  dissolving  1  gram  of  Griibler's  casein  in  a  liter  of  0.1  per  cent  sodium  car- 
bonate.    A  little  chloroform  may  be  added  to  prevent  bacterial  action. 

'The  amount  of  solution  used  may  vary  from  0.1- 1  c.c.  The  measurements  may 
conveniently  be  made  by  means  of  a  i  c.c.  graduated  pipette. 


192  PHYSIOLOGICAL   CHEMISTRY 

do  you  find  the  most  satisfactory  digestion?    How  do  the  results  compare  with 
those  obtained  from  the  similar  series  under  Trypsin,  page  190? 

2.  The  Most  Favorable  Temperature. — (For  this  and  the  following  series  of 
experiments  upon  pancreatic  amylase  use  the  neutral  extract  plus  an  equal  vol- 
imie  of  0.5  per  cent  soditun  carbonate.)  In  each  of  fovu:  tubes  place  2-3  c.c.  of 
alkaline  pancreatic  extract.  Immerse  one  tube  in  cold  water  from  the  faucet, 
keep  a  second  at  room  temperature,  and  place  a  third  on  the  water-bath  at  40°C. 
Boil  the  contents  of  the  fourth  for  a  few  moments,  then  cool  and  also  keep  it  at 
40°C.  Into  each  tube  introduce  2-3  c.c.  of  starch  paste  and  note  the  progress  of 
digestion.  At  the  end  of  one-half  hoiu'  divide  the  contents  of  each  tube  into  two 
parts  and  test  one  part  by  the  iodine  test  and  the  other  part  by  Fehling's  test. 
In  which  tube  do  you  find  the  most  satisfactory  digestion?  How  does  this  result 
compare  with  the  result  obtained  in  the  similar  series  of  experiments  under 
Trypsin  (see  page  190)? 

3.  Influence  of  Bile. — ^Prepare  three  tubes  as  follows : 

(a)  2-3  c.c.  of  pancreatic  extract+2-3  c.c.  of  starch  paste  +  i/2-1  c.c.  of 
bUe. 

(b)  2-3  c.c.  of  pancreatic  extract+2-3  c.c.  of  starch  paste+5  c.c.  of  bile. 

(c)  2-3  c.c.  of  pancreatic  extract+2-3  c.c.  of  starch  paste. 

Shake  the  tubes  thoroughly  and  place  them  in  the  incubator  or  water-bath 
at  40°C.  Note  the  progress  of  digestion  frequently  and  at  the  end  of  a  half -hour 
divide  the  contents  of  each  tube  into  two  parts  and  test  one  part  by  the  iodine 
test  and  the  other  part  by  Fehling's  test.  What  are  yoiu*  conclusions  regarding 
the  influence  of  bile  upon  the  action  of  pancreatic  amylase? 

4.  Digestion  of  Dry  Starch. — To  a  little  dry  starch  in  a  test-tube  add  about 
5  c.c.  of  pancreatic  extract  and  place  the  tube  in  the  incubator  or  water-bath  at 
40°C.  At  the  end  of  a  half-hour  filter  and  test  separate  portions  of  the  filtrate 
by  the  iodine  and  Fehling  tests.  What  do  you  conclude  regarding  the  action  of 
pancreatic  amylase  upon  dry  starch?  Compare  this  result  with  that  obtained  in 
the  similar  experiment  under  Salivary  Digestion  (page  60). 

5.  Digestion  of  Inulin.— To  5  c.c.  of  inulin  solution  in  a  test-tube  add  10  drops 
of  pancreatic  extract  and  place  the  tube  in  the  incubator  or  water-bath  at  40°C. 
After  one-half  hour  test  the  solution  by  Fehling's  test.^  Is  any  reducing  substance 
present?  What  do  you  conclude  regarding  the  digestion  of  inulin  by  pancreatic 
amylase? 

6.  Quantitative  Determination  of  Amylolytic  Activity. — ^Wohlgemuth's 
Method.-  Arrange  a  series  of  test-tubes  with  diminishing  quantities  of  the 
enzyme  solution  under  examination,  introduce  into  each  tube  5  c.c.  of  i  per  cent 
solution  of  soluble  starch^  and  place  each  tube  at  once  in  a  bath  of  ice-water.* 

^  If  the  inulin  solution  gives  a  reduction  before  being  acted  upon  by  the  pancreatic  juice 
it  will  be  necessary  to  determine  the  extent  of  the  original  reduction  by  means  of  a  "check" 
test  (see  p.  47). 

^  Wohlgemuth:  Biochemische  Zeitschrift,  9,  i,  1908. 

'  Kahlbaum's  soluble  starch  is  satisfactory.  In  preparing  the  i  per  cent  solution,  the 
weighed  starch  powder  should  be  dissolved  in  cold  distilled  water  in  a  casserole  and  stirred 
until  a  homogeneous  suspension  is  obtained.  The  mixture  should  then  be  heated,  with  con- 
stant stirring,  untU  it  is  clear.  This  ordinarily  takes  about  8-10  minutes.  A  slightly  opaque 
solution  is  thus  obtained  which  should  be  cooled  and  made  up  to  the  proper  volume  before 
using. 

*  Ordinarily  a  series  of  six  tubes  is  satisfactory,  the  volumes  of  the  enzyme  solution  used 
ranging  from  1  c.c.  to  o.i  c.c.  and  the  measurements  being  made  by  means  of  a  i  c.c.  gradu- 
ated pipette  Each  tube  should  be  placed  in  the  ice-water  bath  as  soon  as  the  starch  solu- 
tion is  introduced.     It  will  be  found  convenient  to  use  a  small  wire  basket  to  hold  the  tubes. 


PANCREATIC   DIGESTION  1 93 

When  all  the  tubes  have  been  prepared  in  this  way  and  placed  in  the  ice-water 
bath  they  are  transferred  to  a  water-bath  or  incubator  and  kept  at  38'C.  for  from 
30  minutes  to  an  hour.'  At  the  end  of  this  digestion  period  the  tubes  are  again 
removed  to  the  bath  of  ice-water  in  order  that  the  action  of  the  enzyme  may 
be  stopped. 

Dilute  the  contents  of  each  tube,  to  within  about  i  2  inch  of  the  top,  with 
water,  add  one  drop  of  a  N  / 10  solution  of  iodine  and  shake  the  tube  and  contents 
thoroughly.  A  series  of  colors  ranging  from  dark  blue  through  bluish  violet  and 
reddish  yellow  to  yellow,  will  be  formed.  -  The  dark  blue  color  shows  the  presence 
of  unchanged  starch,  the  bluish-violet  indicates  a  mixture  of  starch  and  erythro- 
dextrin,  whereas  the  reddish-yellow  signifies  that  erythro dextrin  and  maltose  are 
present  and  the  yellow  solution  denotes  the  complete  transformation  of  starch 
into  maltose.  Examine  the  tubes  carefully  before  a  white  background  and  select 
the  last  tube  in  the  series  which  shows  the  entire  absence  of  all  blue  color,  thus 
indicating  that  the  starch  has  been  completely  transformed  into  dextrins  and 
sugar.  In  case  of  indecision  between  two  tubes,  add  an  extra  drop  of  the  iodine 
solution,  and  observe  them  again,  after  shaking. 

Calculation. — The  amylolytic  activity'  of  a  given  solution  is  expressed  in  terms 
of  the  activity  of  i  c.c.  of  such  a  solution.  For  example,  if  it  is  found  that  0.02 
CO.  of  an  amylolytic  solution,  acting  at  38'C.,  completely  transformed  the  starch 
in  5  c.c.  of  a  I  per  cent  starch  solution  in  30  minutes,  the  amylolytic  activity  of 
such  a  solution  would  be  expressed  as  follows : 

I>3o'=250 

This  indicates  that  i  c.c.  of  the  solution  under  examination  possesses  the  power 
of  completely  digesting  250  c.c.  of  i  per  cent  starch  solution  in  30  minutes  at 
38X. 

"Wohlgemuth  has  suggested  a  slight  alteration  in  the  above  procedure  for  use 
in  the  determination  of  the  amylase  content  of  the  feces. ^  A  modification  of  the 
Wohlgemuth  procedure''  for  this  purpose  is  given  in  the  chapter  on  Feces. 

Experiments  on  Pancreatic  Lipase 

1.  "Litmus-milk"  Test. — Into  each  of  two  test-tubes  introduce  10  c.c.  of  milk 
and  a  small  amount  of  htmus  solution.  To  the  contents  of  one  tube  add  3  c.c. 
of  neutral  pancreatic  extract"^  and  to  the  contents  of  the  other  tube  add  3  c.c.  of 
water  or  of  boiled  neutral  pancreatic  extract.  Keep  the  tubes  at  40'C.  and  note 
any  changes  which  may  occur.     What  is  the  result  and  how  do  you  explain  it? 

2.  Ethyl  Butyrate  Test. — Into  each  of  two  test-tubes  introduce  4  c.c.  of  water, 
2  c.c.  of  ethyl  butyrate,  C3H7COO.C  H,,  and  a  small  amount  of  Utmus  powder. 
To  the  contents  of  one  tube  add  4  c.c.  of  neutral  pancreatic  extract  and  to  the 
contents  of  the  other  tube  add  4  c.c.  of  water  or  of  boiled  neutral  pancreatic  ex- 

'  Longer  digestion  periods  may  be  used  where  it  is  deemed  advisable.  If  exceedingly 
weak  solutions  are  being  investigated,  it  may  be  most  satisfactory  to  permit  the  digestion  to 
extend  over  a  period  of  24  hours. 

*  See  p.  56. 

'  Designated  by  "D"  the  first  letter  of  "diastatic." 

*  Wohlgemuth :  Berliner  klinische  Wochcnschrijt,  47,  92,  1910. 
^  Hawk:  Archives  of  Internal  Medicine,  8,  552,  191 1. 

^  Commercial  pancreatin  may  be  used  in  this  test  if  desired. 


194  PHYSIOLOGICAL    CHEMISTRY 

tract.  Keep  the  tubes  at  40°C.  and  observe  any  change  which  may  occur. 
What  is  the  result  and  how  do  you  explain  it?  Write  the  equation  for  the  reac- 
tion which  has  taken  place. 

Experiments  on  Pancreatic  Rennin 

Prepare  four  test-tubes  as  follows : 

(a)  5  c.c.  of  milk+io  drops  of  neutral  pancreatic  extract. 

(b)  5  c.c.  of  milk +20  drops  of  neutral  pancreatic  extract. 

(c)  5  c.c.  of  milk+io  drops  of  alkaline  pancreatic  extract. 

(d)  5  c.c.  of  milk+20  drops  of  alkaline  pancreatic  extract. 

Place  the  tubes  at  6o°-65°C.  for  a  half  hour  without  shaking.  Note  the 
formation  of  a  clot.^  How  does  the  action  of  pancreatic  rennin  compare  with  the 
action  of  the  gastric  rennin? 

*  This  reaction  will  not  always  succeed,  owing  to  conditions  which  are  not  well  under- 
stood. 


CHAPTER  XI 
INTESTINAL  DIGESTION 

Strictly  speaking,  all  digestive  processes  which  take  place  in  the 
intestine  may  be  classed  under  Intestinal  Digestion.  However,  we  will 
consider  under  Intestinal  Digestion  only  those  digestive  processes 
which  are  brought  about  by  enzymes  which  have  their  origin  in  the  intes- 
tine. The  activities  of  those  enzymes  which  originate  in  the  pancreas 
we  have  considered  in  Chapter  X  under  Pancreatic  Digestion, 

The  enzymes  of  the  intestinal  juice  {succus  entericus)  are  of  great 
importance  to  the  animal  organism.  These  enzymes  include  erepsin, 
sucrose,  maltase,  lactase,  nucleases,  and  enterokinase. 

Erepsin  is  a  proteolytic  enzyme  which  has  the  property  of  acting 
upon  the  proteoses,  peptones,  and  peptides  which  are  formed  through  the 
action  of  trypsin,  and  further  splitting  them  into  amino-acids.  Erepsin 
has  no  power  of  digesting  any  native  proteins  except  caseinogen.  his- 
tones,  and  protamines.  It  possesses  its  greatest  activity  in  an  alkaline 
solution,  although  it  is  slightly  active  in  acid  solution.  An  extract  of  the 
intestinal  erepsin  may  be  prepared  by  treating  the  finely  divided  intes- 
tine of  a  cat,  dog,  or  pig  with  toluol-  or  chloroform-water  and  permitting 
the  mixture  to  stand  with  occasional  shaking  for  24-72  hours.  Enzymes 
similar  to  erepsin  occur  in  various  tissues  of  the  organism. 

In  cases  of  gastric  cancer  a  peptide-splitting  enzyme  is  claimed  to 
be  present  in  the  stomach  contents.  The  glycyl-tryptophane  test  is 
sometimes  used  for  its  detection.  Some  investigators  claim  that  the 
peptide-splitting  power  of  gastric  juice  in  cancer  is  generally  due  to  the 
regurgitation  of  trypsin  or  erepsin  from  the  intestine  or  to  the  presence 
in  the  gastric  contents  of  swallowed  saliva  which  possesses  peptolytic 
power.  The  peptide-splitting  power  of  saliva  may  be  due  to  a  specific 
enzyme  or  to  the  presence  of  bacteria  (see  Glycyltryptophane  Reaction, 
page  199). 

The  three  invertases  sucrase,  maltase,  and  lactase  are  also  important 
enzymes  of  the  intestinal  mucosa.  The  sucrase  acts  upon  sucrose 
and  inverts  it  with  the  formation  of  invert  sugar  (glucose  and  fructose). 
Some  investigators  claim  that  sucrase  is  also  present  in  saliva  and 
gastric  juice.     It  probably  docs  not  exist  normally  in  either  of  these 

195 


196  PHYSIOLOGICAL   CHEMISTRY 

digestive  juices,  however,  and  if  found  owes  its  presence  to  the  excretory 
processes  of  certain  bacteria.  Sucrases  may  also  be  obtained  from 
several  vegetable  sources.  For  investigational  purposes  it  is  ordinarily 
obtained  from  yeast  (see  page  14).  It  exhibits  its  greatest  activity 
in  the  presence  of  a  slight  acidity,  but  if  the  acidity  be  increased  to  any 
extent  the  reaction  is  inhibited. 

Lactase  is  an  enzyme  which  inverts  lactose  with  the  consequent 
formation  of  glucose  and  galactose.  Its  action  is  entirely  analogous, 
in  type,  to  that  of  sucrase.  It  has  apparently  been  proven  that  lactase 
occurs  in  the  intestinal  mucosa  of  the  young  of  all  animals  which  suckle 
their  offspring.^  It  may  also  occur  in  the  intestinal  mucosa  of  certain 
adult  animals  if  such  animals  be  maintained  upon  a  ration  containing 
more  or  less  lactose.  Fischer  and  Armstrong  have  demonstrated  the 
reversible  action^  of  lactase. 

Maltase  possesses  the  power  of  splitting  maltose,  the  end-product 
of  the  digestion  of  starch,  into  glucose.  It  was  first  discovered  in  the 
urine  and  shortly  after  this  time  its  presence  was  noted  in  the  small 
intestine  and  the  saliva.  Corn  is  sometimes  used  as  the  medium  for 
the  preparation  of  the  enzyme  for  experimental  purposes.  It  occurs 
in  corn  in  a  very  active  state.  It  was  in  connection  with  maltase  that 
the  principles  of  the  "reversibility  of  enzyme  action"  were  first 
demonstrated. 

Enierokinase  possesses  the  power  of  "activating"  trypsinogen  see 
Chapters  I  and  X).  In  other  words,  trypsinogen  as  formed  by  the 
pancreas  has  no  proteolytic  power,  but  when  this  inactive  trypsino- 
gen reaches  the  intestine  and  comes  into  contact  with  enterokinase  the 
latter  transforms  it  into  active  trypsin.  Enterokinase  is  not  always 
present  in  the  intestinal  juice  since  it  is  secreted  only  after  the  pan- 
creatic juice  reaches  the  intestine.  It  resembles  the  enzymes  in  that 
its  activity  is  destroyed  by  heat,  but  differs  materially  from  this  class 
of  bodies  in  that  a  certain  quantity  is  capable  of  activating  only  a 
definite  quantity  of  trypsinogen.  It  is,  however,  generally  classified 
as  an  enzyme.  Enterokinase  has  been  detected  in  the  higher  animals, 
and  a  kinase  possessing  similar  properties  has  been  shown  to  be  present 
in  bacteria,  fungi,  impure  fibrin,  lymph  glands,  and  snake-venom. 

The  intestinal  juice  and  the  epithelium  of  the  intestinal  wall  con- 
tain enzymes  capable  of  hydrolyzing  nucleic  acids  and  as  these  acids 
are  not  acted  upon  by  the  gastric  juice  and  probably  not  to  any  great 
extent  by  pure  pancreatic  juice,  the  intestine  apparently  plays  the  chief 
role  in  decomposition  or  digestion  of  these  substances.     At  least  two 

1  Mendel  and  Mitchell:  American  Journal  of  Physiology,  20,  81,  1907. 

2  See  p.  8. 


PANCREATIC   DIGESTION  1 97 

enzymes  take  part  in  this  digestion  process,  one  decomposing  the 
nucleic  acid  with  formation  of  simple  nucleotides  containing  a  single 
radical  each  of  phosphoric  acid,  carbohydrate  and  base  (see  chapter 
on  Nucleic  Acids).  This  enzyme  may  be  called  nudeicacidase.  An- 
other enzyme  present  in  the  intestine  and  intestinal  juice  decomposes 
these  nucleotides  with  the  liberation  of  phosphoric  acid.  This  enzyme 
may  be  called  ■nucleotidase  or  phosplwnuclcase.  The  intestinal  mucosa 
also  decomposes  many  other  organic  phosphorus  compounds  with  libera- 
tion of  their  phosphoric  acid.^  Thus  glycero-phosphoric  acid  and 
hexose-phosphoric  acid  as  well  as  phosphoproteins  are  split  in  a  similar 
manner,  the  phosphoric  acid  they  contain  thus  being  absorbed  in  the 
free  form. 

General  Experiments  on  Intestinal  Digestion 

Demonstration  of  Enterokinase. — Trypsinogen  may  be  activated 
by  enterokinase.  This  activation  occurs  normally  in  the  intestine. 
Calcium  salts  also  bring  about  a  similar  activation  of  the  trypsinogen. 

Procedure. — Prepare  an  extract  of  trypsinogen  by  grinding  10  grams  of  the 
fresh,  fat-free  pancreas  of  the  pig  with  a  Uttle  sand.  Gradually  add  100  c.c. 
of  water  during  the  grinding  process.     Strain  through  cheese  cloth. 

Prepare  an  extract  of  enterokinase  by  grinding  5  grams  of  fresh,  fat-free 
duodenal  mucosa-  of  the  pig  with  a  Uttle  sand.  Gradually  add  50  c.c.  of  water 
during  the  grinding  process.     Strain  through  cheese  cloth. 

Prepare  the  following  series  of  tubes : 

(a)  10  c.c.  pancreas  extract+5  c.c.  water. 

(b)  10  c.c.  pancreas  extract+5  c.c.  duodenal  extract. 

(c)  5  c.c.  duodenal  extract +10  c.c.  water. 

(d)  10  c.c.  pancreas  extract+5  cc.  duodenal  extract. 

(e)  10  c.c.  pancreas  extract+5  c.c.  duodenal  extract  (boiled^*. 

(f)  10  c.c.  pancreas  extract+5  c.c.  of  4  per  cent  calcium  chloride. 

Boil  the  contents  of  tube  (d)  for  five  minutes  and  cool  to  40°C.  Keep  all  six  tubes 
at  40°C.  for  20  minutes. 

To  each  tube  add  5  c.c.  of  10  per  cent  sodium  carbonate  and  mix  the  contents 
thoroughly  and  immediately.  Introduce  into  each  tube  the  same  quantity  (size 
of  a  pea)  of  fresh  fibrin.  Shake  the  tubes  and  place  them  at  40°C.  Observe  the 
tubes  frequently  for  one  hour  to  note  digestive  changes.  Tubes  (b)  and  (f) 
should  show  most  rapid  digestion.     Why? 

Experiments  on  Intestinal  Nucleases 

I.  Preparation  of  Intestinal  Extract. — Wash  thoroughly  100  grams  of  pig's 
intestine  and  run  through  a  meat  chopper  several  times.  Introduce  into  a  500 
c.c.  mixing  cyUnder  and  add  normal  salt  solution  to  make  500  c.c.     Allow  to 

^  Plimmer:  Biochem.  J.,  7,  43,  1913. 

^  The  dried  mucosa  may  be  substituted  if  desired. 


198  PHYSIOLOGICAL    CHEMISTRY 

stand  for  6-24  hours  at  room  temperatxire,  shaking  occasionally,  toluol  being 
added  as  a  preservative.     Strain  and  filter. 

2.  Demonstration  of  Intestinal  Nucleases. — Prepare  a  2  per  cent  solution 
of  yeast  nucleic  acid  put  in  solution  with  the  aid  of  just  sufficient  NaOH  solution 
to  make  the  resulting  mixture  neutral  to  Utmus.  To  each  of  two  large  test-tubes 
add  20  c.c.  of  the  intestinal  extract  prepared  as  above.  Boil  one  for  one  to  two 
minutes.  To  each  tube  then  add  10  c.c.  of  the  2  per  cent  nucleic  acid  solution. 
Add  2-3  c.c.  each  of  toluol  and  chloroform  to  each  mixture.  Keep  at  38°C.  for 
24  hours. 

Heat  the  tubes  to  boiling  in  a  water-bath  to  coagulate  protein.  Add  5  c.c.  of 
5  per  cent  HCl  and  allow  to  stand  for  one  hour.  This  precipitates  any  unchanged 
nucleic  acid.  Filter  and  take  aUquots  of  the  filtrate  (about  20  c.c).  Precipitate 
the  phosphate  from  each  mixture  by  adding  5  c.c.  of  magnesia  mixture  and  5  c.c. 
of  ammonia.  Allow  to  stand  over  night.  A  heavy  precipitate  of  magnesium 
ammonium  phosphate  should  be  found  in  the  test  experiment  indicating  that  the 
phosphoric  acid  of  the  nucleic  acid  had  been  Uberated  by  the  nucleotidase  of  the 
intestinal  extract.     The  control  should  show  only  a  sUght  precipitate. 

If  desired  the  phosphorus  of  the  precipitates  may  be  determined  quantitatively 
by  dissolving  in  2  per  cent  HNO3,  precipitating  as  the  phosphomolybdate  and 
determining  volumetrically  according  to  the  Neumann  procedure  (see  554). 


Experiments  on  Erepsin 

1.  Preparation  of  Erepsin. — Grind  the  mucous  membrane  of  the  small  intes- 
tine of  a  cat,  dog,  or  pig  with  sand  in  a  mortar.  Treat  the  finely  divided  membrane 
with  toluol-  or  chloroform-water  and  permit  the  mixture  to  stand,  with  occasional 
shaking,  for  24-72  hours.  ^  Filter  the  extract  thus  prepared  through  cotton  and 
use  the  filtrate  in  the  following  experiment. 

2.  Demonstration  of  Erepsin. — To  about  5  c.c.  of  a  i  per  cent  solution  of 
Witte's  peptone  in  a  test-tube  add  about  i  c.c.  of  the  erepsin  extract  prepared  as 
described  above  and  make  the  mixture  slightly  alkaUne  (o.i  per  cent)  with  sodiimi 
carbonate.  Prepare  a  second  tube  containing  a  like  amount  of  peptone  solution 
but  boil  the  erepsin  extract  before  introducing  it.  Place  the  two  tubes  at  38°C. 
for  two  to  three  days.  At  the  end  of  that  period  heat  the  contents  of  each  tube 
to  boiling,  filter  and  try  the  biuret  test  on  each  filtrate.  In  making  these  tests 
care  should  be  taken  to  use  Uke  amounts  of  filtrate,  potassium  hydroxide  and 
copper  sulphate  in  each  test  in  order  that  the  drawing  of  correct  conclusions  may 
be  faciUtated.  The  contents  of  the  tube  which  contained  the  boiled  extract 
should  show  a  deep  pink  color  with  the  biuret  test,  due  to  the  peptone  still  present. 
On  the  other  hand,  the  biuret  test  upon  the  contents  of  the  tube  containing  the 
unboiled  extract  should  be  negative  or  exhibit,  at  the  most,  a  faint  pink  or  blue 
color,  signifying  that  the  peptone,  through  the  influence  of  the  erepsin,  has  been 
transformed,  in  great  part  at  least,  into  amino-acids  which  do  not  respond  to  the 
biuret  test- 

1  The  enzyme  may  also  be  extracted  by  means  of  glycerol  or  alkaline  "physiological"  salt 
solution  if  desired. 

^  Strictly  speaking,  this  erepsin  demonstration  is  not  adequate  unless  a  control  test 
is  made  with  native  protein  (except  casein,  histones  and  protamines)  to  show  that  the 
extract  is  trypsin-free  and  digests  peptone  but  not  native  protein. 


INTESTINAL   DIGESTION  1 99 

3.  The  Glycyl-tryptophane  Reaction. — The  dipeptide  glycyl -tryptophane  1 
may  be  used  in  place  of  the  peptone  solution  for  the  demonstration  of  erepsin. 
It  is  claimed  to  be  of  service  in  the  diagnosis  of  gastric  cancer.  It  is  claimed  that 
a  peptide -spUtting  enzyme  (erepsin)  is  present  in  the  stomach  contents  of  indi- 
viduals suffering  from  cancer  of  the  stomach,  whereas  the  stomach  contents  of 
normal  individuals  contains  no  such  enzyme.  The  glycyl-tryptophane  test,  there- 
fore, may  sometimes  furnish  a  means  of  aiding  in  the  diagnosis  of  this  disorder. 
As  applied  to  stomach  contents,  the  test  is  as  follows :-  Introduce  about  10  c.c.  of 
the  filtrate  from  the  stomach  contents  into  a  test-tube,  add  a  little  glycyl-trypto- 
phane and  a  layer  of  toluol,  and  place  the  tube  in  an  incubator  at  38'C.  for  24 
hours.  At  the  end  of  this  time  by  means  of  a  pipette  transfer  2-3  c.c.  of  the  fluid 
from  beneath  the  toluol  to  a  test-tube,  add  a  few  drops  of  3  per  cent  acetic  acid 
and  carefully  introduce  bromine  vapors.  Shake  the  tube  and  note  the  production 
of  a  red  color  if  tryptophane  is  present.  The  tryptophane  has,  of  course,  been 
liberated  from  the  peptide  through  the  action  of  the  peptide -spUtting  enzyme 
(erepsin)  elaborated  by  the  cancer  tissue. 

If  an  excess  of  bromine  is  added  the  color  will  vanish.  If  no  rose  color  is 
noted,  add  more  bromine  vapors  carefully  with  shaking  until  further  addition  of 
the  vapors  causes  the  production  of  a  yellowish  color.  This  indicates  an  excess 
of  bromine  and  constitutes  a  negative  test.  Occasionally  the  rose  color  indicating 
a  positive  test  is  so  transitory  as  to  escape  detection  unless  the  test  be  very  care- 
fully performed. 

Several  fallacies  have  been  pointed  out  in  connection  with  this  test. 
In  the  first  place  the  regurgitation  of  duodenal  contents  through  the 
pylorus  might  insure  the  presence  in  the  stomach  of  erepsin  and  trypsin 
either  of  which  possesses  peptide-splitting  power.  It  has  also  been 
claimed  that  saliva  contains  an  enzyme  capable  of  splitting  glycyl- 
tryptophane.  Doubt  has,  however,  been  cast  upon  the  dipeptide- 
splitting  agent  of  the  saliva  by  Smithies^  and  by  Jacque  and  Woodyatt,^ 
who  point  to  bacteria  as  the  peptolytic  agents.  In  any  event  saliva 
contains  something  which  is  capable  of  splitting  the  glycyltryptophan, 
thus  making  the  entrance  of  saliva  into  the  stomach  an  important 
source  of  error,  so  far  as  the  utility  of  this  test  is  concerned,  as  a  diagnos- 
tic aid.  Bacteria  may,  of  course,  be  removed  from  the  gastric  juice  by 
passing  the  fluid  through  an  efTective  filter. 

Experiments  on  Invert ases^ 

I.  Preparation  of  an  Extract  of  Sucrase. — Treat  the  finely  divided  epi- 
thelium of  the  small  intestine  of  a  dog,  pig,  rat,  rabbit,  or  hen  with  about  3 
voltimes  of  a  2  per  cent  solution  of  sodium  fluoride  and  permit  the  mixture  to 

^  This  dipeptide  is  sold  commercially  under  the  name  "Ferment  Diagnosticon." 
-  Neubauer  and  Fischer:  Deulsclies  Archiv  f.  klinische  Medizin,  97,  499,  1909. 
^  Smithies:  Arch.  Int.  Med.,  10,  521,  191 2. 

*  Jacque  and  Woodyatt:  Arch.  Int.  Med.,  Dec,  191 2,  p.  560. 

*  "The  Inverting  Enzymes  of  the  Alimentary  Tract,"  Mendel  and  Mitchell:  American 
Journal  of  Physiology,  20,  81,  1907-08. 


200  PHYSIOLOGICAL    CHEMISTRY 

stand  at  room  temperature  for  24  hours.     Strain  the  extract  through  cloth  or 
absorbent  cotton  and  use  the  strained  material  in  the  following  demonstration. 

2.  Demonstration  of  Sucrase. — To  about  5  c.c.  of  a  i  per  cent  solution  of 
sucrose,  in  a  test-tube,  add  about  i  c.c.  of  a  2  per  cent  sodium  fluoride  intes- 
tinal extract,  prepared  as  described  above.  Prepare  a  control  tube  in  which  the 
intestinal  extract  is  boiled  before  being  added  to  the  sugar  solution.  Place  the 
two  tubes  at  38°C.  for  two  hours.  ^  Heat  the  mixture  to  boiUng  to  coagulate  the 
protein  material,  filter,  and  test  the  filtrate  by  Fehling's  test  (see  page  26). 
The  tube  containing  the  boiled  extract  should  give  no  response  to  Fehling's  test, 
whereas  the  tube  containing  the  unboiled  extract  should  reduce  the  Fehling's 
solution.  This  reduction  is  due  to  the  formation  of  invert  sugar  (see  page  41 ) 
from  the  sucrose  through  the  action  of  the  enzyme  sucrase  which  is  present  in 
the  intestinal  epithelium. 

For  preparation  and  demonstration  of  Vegetable  Sucrase  see  Chapter  I. 

3.  Preparation  of  an  Extract  of  Lactase. — Treat  the  finely  divided  epithelium 
of  the  small  intestine  of  a  kitten,  puppy,  or  pig  embryo  with  about  3  volumes  of  a 
2  per  cent  solution  of  sodium  fluoride  and  permit  the  mixture  to  stand  at  room 
temperature  for  24  hours.  Strain  the  extract  through  cloth  or  absorbent  cotton 
and  use  the  strained  material  in  the  following  demonstration. 

4.  Demonstration  of  Lactase.  ^ — To  about  5  c.c.  of  a  i  per  cent  solution  of 
lactose  in  a  test-tube  add  about  i  c.c.  of  a  toluol-water  or  a  2  per  cent  sodium 
fluoride  extract  of  the  first  part  of  the  small  intestine^  of  a  kitten,  puppy,  or  pig 
embryo  prepared  as  described  above.  Prepare  a  control  tube  in  which  the 
intestinal  extract  is  boiled  before  being  added  to  the  sugar  solution.  Place  the 
two  tubes  at  38°C.  for  24  hours.  At  the  end  of  this  period  add  i  c.c.  of  the  diges- 
tion mixture  to  5  c.c.  of  Barfoed's  reagent*  and  place  the  tubes  in  a  boiUng  water- 
bath.^  Examine  the  tubes  at  the  end  of  three  minutes  against  a  black  back- 
ground in  a  good  Ught.  If  no  cuprous  oxide  is  visible  replace  the  tubes  and 
repeat  the  examination  at  the  end  of  the  fourth  and  fifth  minutes.  If  no  reduc- 
tion is  then  observed  permit  the  tubes  to  stand  at  room  temperature  for  5-10 
minutes  and  examine  again.  "^ 

It  has  been  determined  that  disaccharide  solutions  will  not  reduce  Barfoed's 
reagent  until  after  they  have  been  heated  for  9-10  minutes  on  a  boiUng  water- 
bath  in  contact  with  the  reagent.^  Therefore  in  the  above  test,  if  the  tube  con- 
taining the  unboiled  extract  exhibits  any  reduction  after  being  heated  as  indicated, 
for  a  period  of  five  minutes  or  less,  and  the  control  tube  containing  boiled  extract 
shows  no  reduction,  it  may  be  concluded  that  lactase  was  present  in  the  intestinal 
extract.  ** 

^  If  a  positive  result  is  not  obtained  in  this  time  permit  the  digestion  to  proceed  for  a 
longer  period. 

-  Roaf:  Bio-Chemical  Journal,  3,  182,  1908. 
'  Duodenum  and  first  part  of  jejunum. 

*  To  4.5  grams  of  neutral  crystallized  copper  acetate  in  900  c.c.  of  water  add  0.6  c.c.  of 
glacial  acetic  acid  and  make  the  total  volume  of  the  solution  i  liter. 

^  Care  should  be  taken  to  see  that  the  water  in  the  bath  reaches  at  least  to  the  upper 
level  of  the  contents  of  the  tubes. 

*  Sometimes  the  drawing  of  conclusions  is  facilitated  by  pouring  the  mixture  from  the 
tube  and  examining  the  bottom  of  the  tube  for  adherent  cuprous  oxide. 

'  The  heating  for  9-10  minutes  is  sufficient  to  transform  the  disaccharide  into  mono- 
saccharide. 

8  The  reduction  would,  of  course,  be  due  to  the  action  of  the  glucose  and  galactose 
which  had  been  formed  from  the  lactose  through  the  action  of  the  enzyme  lactase. 


INTESTINAL   DIGESTION  20I 

5.  Preparation  of  an  Extract  of  Maltase.-  Treat  the  finely  divided  epithelium 
of  the  small  intestine  of  a  cat,  kitten,  or  pig  fembryo  or  adult)  with  about  3 
volumes  of  a  2  per  cent  solution  of  sodium  fluoride  and  permit  the  mixture  to 
stand  at  room  temperature  for  24  hours.  Strain  the  extract  through  cloth  and 
use  the  strained  material  in  the  following  demonstration. 

6.  Demonstration  of  Maltase. — Proceed  exactly  as  indicated  in  the  demon- 
stration of  lactase,  p.  200,  except  that  a  i  per  cent  solution  of  maltose  is  sub- 
stituted for  the  lactose  solution.  The  extract  used  may  be  prepared  from  the 
upper  part  of  the  intestine  of  a  cat,  kitten,  or  pig  (embryo  or  adult).  In  the  case 
of  lactase,  as  indicated,  the  intestine  used  should  be  that  of  a  kitten,  puppy,  or 
pig  (embryo). 


CHAPTER  XII 
BILE 

The  bile  is  secreted  continuously  by  the  liver  and  passes  into  the 
intestine  through  the  common  bile  duct  which  opens  near  the  pylorus. 
Bile  is  not  secreted  continuously  into  the  intestine.  In  a  fasting  animal 
no  bile  enters  the  intestine,  but  when  food  is  taken  the  bile  begins  to 
flow;  the  length  of  time  elapsing  between  the  ingestion  of  the  food  and 
the  secretion  of  the  bile  as  well  as  the  qualitative  and  quantitative  char- 
acteristics of  the  secretion  depending  upon  the  nature  of  the  food  ingested. 
Fats,  the  extractives  of  meat  and  the  protein  end-products  of  gas- 
tric digestion  (proteoses  and  peptones),  cause  a  copious  secretion  of  bile, 
whereas  such  substances  as  water,  acids  and  boiled  starch  paste  fail 
to  do  so.  In  general  a  rich  protein  diet  is  supposed  to  increase  the 
amount  of  bile  secreted,  whereas  a  carbohydrate  diet  would  cause  a 
much  less  decided  increase  and  might  even  tend  to  decrease  the  amount. 
It  has  been  demonstrated  by  Bayliss  and  Starling  that  the  secretion  of 
bile  is  under  the  control  of  the  same  mechanism  that  regulates  the  flow 
of  pancreatic  juice  (see  page  185).  In  other  words,  the  hydrochloric 
acid  of  the  chyme,  as  it  enters  the  duodenum  transforms  prosecretin 
into  secretin  and  this  in  turn  enters  the  circulation,  is  carried  to  the 
liver,  and  stimulates  the  bile-forming  mechanism  to  increased  activity. 

We  may  look  upon  the  bile  as  an  excretion  as  well  as  a  secretion.  In 
the  fulfillment  of  its  excretory  function  it  passes  such  bodies  as  lecithin, 
metallic  substances,  cholesterol,  and  the  decomposition  products  of 
hemoglobin  into  the  intestine  and  in  this  way  aids  in  removing  them 
from  the  organism.  The  bile  assists  materially  in  the  absorption  of 
fats  from  the  intestine  by  its  solvent  action  on  the  fatty  acids  formed 
by  the  action  of  the  pancreatic  juice. 

The  bile  is  a  ropy,  viscid  substance  which  is  usually  alkaline  in  reac- 
tion to  litmus,  ^  and  ordinarily  possesses  a  decidedly  bitter  taste.  It  varies 
in  color  in  the  different  animals,  the  principal  variations  being  yellow, 
brown  and  green.  Fresh  human  bile  from  the  living  organism  ordi- 
narily has  a  green  or  golden-yellow  color.  Post-mortem  bile  is  variable 
in  color.  It  is  very  difficult  to  determine  accurately  the  amount  of 
normal  bile  secreted  during  any  given  period.     For  an  adult  man  it 

*  It  does  not  contain  more  than  a  slight  excess  of  hydroxyl  ions,  however. 

202 


BILE 


203 


has  been  variously  estimated  at  from  500  c.c.  to  iioo  c.c.  for  24  hours. 
The  specific  gravity  of  the  bile  varies  between  i.oio  and  1.040,  and 
the  freezing-point  is  about  —  o.56°C.  As  secreted  by  the  liver,  the 
bile  is  a  clear,  limpid  fluid  which  contains  a  relatively  low  content  of 
solid  matter.  Such  bile  would  have  a  specific  gravity  of  approximately 
I.OIO.  After  it  reaches  the  gall-bladder,  however,  it  becomes  mixed 
with  mucous  material  from  the  walls  of  the  gall-bladder,*  and  this  proc- 
ess coupled  with  the  continuous  absorption  of  water  from  the  bile  has 
a  tendency  to  concentrate  the  secretion.  Therefore  the  bile  as  we  find  it 
in  the  gall-bladder  ordinarily  possesses  a  higher  specific  gravity  than 
that  of  the  freshly  secreted  fluid.  The  specific  gravity  under  these 
conditions  may  run  as  high  as  1.040. 

The  principal  constituents  of  the  bile  are  the  salts  of  the  bile  acids, 
bile  pigments,  neutral  fats,  lecithin,  phosphatides,  nucleo protein,  mucin, 
and  cholesterol,  besides  the  salts  of  iron,  copper,  calcium,  and  magnesium. 
Zinc  has  also  frequently  been  found  in  traces. 

The  quantitative  composition  of  bile  varies  according  to  the  source 
of  the  bile,  i.e.,  whether  the  bile  for  analysis  is  obtained  from  the  gall- 

QUANTITATIVE  COMPOSITION  OF  BILEi 
(Parts  per  1000) 


Constituent 

Bladder  bile, 
Hammartsen- 

Biliary  fistula, 
Rosenbloom' 

Water 

Solids 

'                 829.7 

170.3 

970.2 
39-8 

Bile  salts 

Q7    0 

10  I 

Mucin  and  pigments 

J.I  .  0 

4.86 

Cholesterol 

9-9 

2.6l5 

Fat 

I                      1-9 

6.85 

Soaps 

II. 2* 

1 

2.6 

Lecithin 

'                      2.2                    ' 

6.42 

Inorganic  matter 

S.I 

9.2 

Fatty  acids 

Included  under  "soaps" 

1.2 

*  For  other  analyses  see  Czylharz,  Fuchs  and  v.  Fiirth:  Block.  Zeit.,  49,  120,    1913. 

*Hammarsten:  Pincussohn's  Med.-Chem.  Lab.  Hilfsbuch,  Leipzig,  191 2,  p.  388. 
'Rosenbloom:  Jour.  Biol.  Chcin.,  14,  241,  1913. 

*  Includes  fatty  acids. 

*  Includes  cholesterol  esters. 


204  PHYSIOLOGICAL    CHEMISTRY 

bladder  or  by  means  of  a  fistula  before  it  reaches  the  gall-bladder. 
The  variation  in  the  composition  of  these  two  types  of  bile  is  shown  in 
the  preceding  selected  analyses: 

The  bile  acids,  which  are  elaborated  exclusively  by  the  hepatic 
cells,  may  be  divided  into  two  groups,  the  glycocholic  acid  group  and 
the  taurocholic  acid  group.  In  human  bile  glycocholic  acid  predomi- 
nates, while  taurocholic  acid  is  the  more  abundant  in  the  bile  of  car- 
nivora.  The  bile  acids  are  conjugate  amino-acids,  the  glycocholic 
acid  yielding  glycocoll, 

CH2NH2 

COOH, 

and  cholic  acid  upon  decomposition,  whereas  taurocholic  acid  give 
rise  to  taurine, 

CH2NH2 

CH2SO2OH, 

and  cholic  acid  under  like  conditions.  Glycocholic  acid  contains  some 
nitrogen  but  no  sulphur,  whereas  taurocholic  acid  contains  both  these 
elements.  The  sulphur  of  the  taurocholic  acid  is  present  in  the  taurine 
(amino-ethyl-sulphonic-acid) ,  of  which  it  is  a  characteristic  constituent. 
There  are  several  varieties  of  cholic  acid  and  therefore  we  have  several 
forms  of  glycocholic  and  taurocholic  acids,  the  variation  in  constitution 
depending  upon  the  nature  of  the  cholic  acid  which  enters  into  the  com- 
bination. The  bile  acids  are  present  in  the  bile  as  salts  of  one  of  the 
alkalis,  generally  sodium.  The  sodium  glycocholate  and  sodium  tau- 
rocholate  may  be  isolated  in  crystalline  form,  either  as  balls  or  rosettes 
of  fine  needles  or  in  the  form  of  prisms  having  ordinarily  four  or  six 
sides  (Fig.  55,  page  205) .  The  salts  of  the  bile  acids  are  dextro-rotatory. 
Among  other  properties  these  salts  have  the  power  of  holding  the 
cholesterol  and  lecithin  of  the  bile  in  solution. 

Hammarsten  has  demonstrated  a  third  group  of  bile  acids  in  the 
bile  of  the  shark.  This  same  group  very  probably  occurs  in  certain 
other  animals  also.  These  acids  are  very  rich  in  sulphur  and  resemble 
etheral  sulphuric  acids  inasmuch  as  upon  treatment  with  boiling  hydro- 
chloric acid  they  yield  sulphuric  acid. 

The  bile  pigments  are  important  and  interesting  biliary  constitu- 
ents. The  following  have  been  isolated:  bilirubin,  biliverdin,  bili- 
fuscin,  biliprasin,  bilihumin,  bilicyanin,  choleprasin,  and  choletelin.  Of 
these,  bilirubin  and  biliverdin  are  the  most  important  and  predominate 
in  normal  bile.  The  colors  possessed  by  the  various  varieties  of  normal 
bile  are  due  almost  entirely  to  these  two  pigments,  the  biliverdin  being 


BILE 


20: 


the  predominant  pigment  in  greenish  bile  and  the  bilirubin  being  the 
principal  pigment  in  lighter  colored  bile.  The  pigments,  other  than 
the  two  just  mentioned,  have  been  found  almost  exclusively  in  biliary 
calculi  or  in  altered  bile  obtained  at  post-mortem  examinations. 

Bilirubin,  which  is  perhaps  the  most  important  of  the  bile  pigments, 
is  apparently  derived  from  the  blood  pigment,  the  iron  freed  in  the 


Fig.  55. — Bile  Salts. 

process  being  held  in  the  liver.  Bilirubin  has  the  same  percentage  com- 
position as  hematoporphyrin,  which  may  be  produced  from  hematin. 
It  is  a  specific  product  of  the  liver  cells,  but  may  also  be  formed  in  other 
parts  of  the  body.  The  pigment  may  be  isolated  in  the  form  of  a 
reddish-yellow  powder  or  may  be  obtained  in  part,  in  the  form  of  reddish- 


# 


t 


Fig.  56. — Bilirubin  (Hzmatoidin).     (Ogden.) 

yellow  rhombic  plates  (Fig.  56)  upon  the  spontaneous  evaporation 
of  its  chloroform  solution.  The  crystalline  form  of  bilirubin  is 
practically  the  same  as  that  of  hematoidin.  It  is  easily  soluble  in 
chloroform,  somewhat  less  soluble  in  alcohol  and  only  slightly  soluble 
in  ether  and  benzene.     Bilirubin  has  the  power  of  combining  with 


206  PHYSIOLOGICAL   CHEMISTRY 

certain  metals,  particularly  calcium,  to  form  combinations  which  are  no 
longer  soluble  in  the  solvents  of  the  unaltered  pigment.  Upon  long 
standing  in  contact  with  the  air,  the  reddish-yellow  bilirubin  is  oxidized 
with  the  formation  of  the  green  biliverdin.  Bilirubin  occurs  in  animal 
fluids  as  soluble  bilirubin-alkali. 

Solutions  of  bilirubin  exhibit  no  absorption  bands.  If  an  ammonia- 
cal  solution  of  bilirubin-alkali  in  water  is  treated  with  a  solution  of  zinc 
chloride,  however,  it  shows  bands  similar  to  those  of  bilicyanin  (Absorp- 
tion Spectra,  Plate  II),  the  two  bands  between  C  and  D  being  rather 
well  defined. 

Biliverdin  is  particularly  abundant  in  the  bile  of  herbivora.  It  is 
soluble  in  alcohol  and  glacial  acetic  acid  and  insoluble  in  water,  chloro- 
form, and  ether.  Biliverdin  in  formed  from  bilirubin  upon  oxidation. 
It  is  an  amorphous  substance,  and  in  this  differs  from  bilirubin  which 
may  be  at  least  partly  crystallized  under  proper  conditions.  Biliverdin 
may  be  obtained  in  the  form  of  a  green  powder.  In  common  with 
bilirubin,  it  may  be  converted  into  hydrobilirubin  by  nascent  hydrogen. 

The  neutral  solution  of  bilicyanin  or  cholecyanin  is  bluish  green  or 
steel  blue  and  possesses  a  blue  fluorescence,  the  alkaline  solution  is 
green  with  no  appreciable  fluorescence,  and  the  strongly  acid  solution  is 
violet  blue.  The  alkaline  solution  exhibits  three  absorption  bands,  the 
first  a  dark,  well-defined  band  between  C  and  D,  somewhat  nearer  C; 
the  second  a  less  sharply  defined  band  extending  across  D  and  the  third 
a  rather  faint  band  between  E  and  F,  near  E  (Absorption  Spectra,  Plate 
II).  The  strongly  acid  solution  exhibits  two  absorption  bands,  both 
lying  between  C  and  E  and  separated  by  a  narrow  space  near  D.  A 
third  band,  exceedingly  faint,  may  ordinarily  be  seen  between  b  and  F. 

Bile  pigments  are  converted  into  urobilinogen  (urobilin)  in  the  intes- 
tine. This  is  absorbed,  carried  to  the  liver  and  reconverted  into  bile 
pigment.  In  diseases  of  the  liver  the  liver  cell  loses  the  capacity  to 
convert  the  urobilinogen  and  this  is  then  excreted  in  the  urine.  The 
presence  of  urobilinogen  in  urine,  therefore,  may  be  considered  as  an 
index  oi  functional  liver  incapacity.^ 

Biliary  calculi,  otherwise  designated  as  biliary  concretions  or  gall 
stones,  are  frequently  formed  in  the  gall-bladder.  These  deposits  may 
be  divided  into  three  classes,  cholesterol  calculi,  pigment  calculi,  and 
calculi  made  up  almost  entirely  of  inorganic  material.  This  last  class 
of  calculus  is  formed  principally  of  the  carbonate  and  phosphate  of 
calcium  and  is  rarely  found  in  man  although  quite  common  to  cattle. 
The  pigment  calculus  is  also  found  in  cattle,  but  is  more  common  to 
man  than  the  inorganic  calculus.     This  pigment  calculus  ordinarily 

1  Rowntree,  Hurwitz  and  Bloomfield:  Johns  Hopkins  Hospital  Bulletin,  Nov.,  1913. 


BILE  207 

consists  principally  of  bilirubin  in  combination  with  calcium;  biliverdin 
is  sometimes  found  in  small  amount.  The  cholesterol  calculus  is  the  one 
found  most  frequently  in  man.  These  may  be  formed  almost  entirely 
of  cholesterol,  in  which  event  the  color  of  the  calculus  is  very  light,  or 
they  may  contain  more  or  less  pigment  and  inorganic  matter  mixed 
with  the  cholesterol,  which  tends  to  give  us  calculi  of  various  colors. 
For  discussion  of  cholesterol  see  page  355. 

Experiments  on  Bile 

1.  Reaction. — Test  the  reaction  of  fresh  ox  bile  to  litmus,  phenolphthalein  and 
Congo  red. 

2.  Nucleprotein. — Acidify  a  small  amount  of  bile  with  dilute  acetic  acid.  A 
precipitate  of  nucleoprotein  forms.  Bile  acids  will  also  precipitate  here  under 
proper  conditions  of  acidity. 

3.  Inorganic  Constituents. — Test  for  chlorides,  sulphates,  and  phosphates 
(see  page  59). 

4.  Tests  for  Bile  Pigments.— Practically  all  of  these  tests  for  bile 
pigments  are  based  on  the  oxidation  of  the  pigment,  by  a  variety  of 
reagents,  with  the  formation  of  a  series  of  colored  derivatives,  e.g., 
biliverdin  (green),  bilicyanin  (blue),  choletelin  (yellow). 

(a)  Gmelin's  Test. — To  about  5  c.c.  of  concentrated  nitric  acid  in  a  test-tube 
add  2-3  c.c.  of  diluted  bile  carefully  so  that  the  two  fluids  do  not  mix.  At  the 
point  of  contact  note  the  various  colored  rings,  green,  blue,  violet,  red  and  reddish 
yellow.     Repeat  this  test  with  different  dilutions  of  bile  and  observe  its  deUcacy. 

(b)  Rosenbach's  Modification  of  Gmelin's  Test. — Filter  5  c.c.  of  diluted  bile 
through  a  small  filter  paper.  Introduce  a  drop  of  concentrated  nitric  acid  into 
the  cone  of  the  paper  and  note  the  succession  of  colors  as  given  in  Gmelin's  test. 

(a)  Nakayama's  Reaction. — To  5  c.c.  of  diluted  bile  in  a  test-tube  add  an  equal 
volume  of  a  10  per  cent  solution  of  barium  chloride,  centrifugate  the  mixture,  pour 
off  the  supernatant  fluid,  and  heat  the  precipitate  with  2  c.c.  of  Nakayama's  reagent.^ 
In  the  presence  of  bile  pigments  the  solution  assumes  a  blue  or  green  color. 

(d)  Huppert's  Reaction.^ — Thoroughly  shake  equal  volumes  of  undiluted  bile 
and  milk  of  lime  in  a  test-tube.  The  pigments  unite  with  the  calcium  and  are  pre- 
cipitated. Filter  off  the  precipitate,  wash  it  with  water,  and  transfer  to  a  small 
beaker.  Add  alcohol  acidified  slightly  with  hydrochloric  acid  and  warm  upon  a 
water-bath  until  the  solution  becomes  colored  an  emerald  green. 

In  examining  urine  for  bile  pigments,  according  to  Steensma,  this  procedure 
may  give  negative  results  even  in  the  presence  of  the  pigments,  owing  to  the  fact 
that  the  acid-alcohol  is  not  a  sufllciently  strong  oxidizing  agent.  He  therefore 
suggests  the  addition  of  a  drop  of  a  0.5  per  cent  solution  of  sodium  nitrite  to  the 
acid-alcohol  mixture  before  warming  on  the  water-bath.     Try  this  modification  also. 

(e)  Hammarsten's  Reaction.— To  about  5  cc.  of  Hammarsten's  reagent-  in  a 

1  Prepared  by  combining  99  c.c.  of  alcohol  and  i  c.c.  of  fuming  hydrochloric  acid  con- 
taining 4  grams  of  ferric  chloride  per  liter. 

^  Hammarsten's  reagent  is  made  by  mi.\ing  i  volume  of  25  per  cent  nitric  acid  and  19 
volumes  of  25  per  cent  hydrochloric  acid  and  then  adding  i  volume  of  this  acid  mbtture 
to  4  volumes  of  95  per  cent  alcohol. 


2o8  PHYSIOLOGICAL   CHEMISTRY 

small  evaporating  dish  add  a  few  drops  of  diluted  bile.  A  green  color  is  produced. 
If  more  of  the  reagent  is  now  added  the  play  of  colors  as  observed  in  Gmelin's  test 
may  be  obtained. 

(f)  Smith's  Test. — To  2-3  c.c.  of  diluted  bile  in  a  test-tube  add  carefully  about 
5  c.c.  of  dilute  tincture  of  iodine  (i  :  10)  so  that  the  fluids  do  not  mix.  A  play  of 
colors,  green,  blue  and  violet,  is  observed.  In  making  this  test  upon  the  urine  ordin- 
arily only  the  green  color  is  observed. 

(g)  Salkowski-Schipper's  Reaction. — To  10  c.c.  of  diluted  bile  in  a  test-tube 
add  5  drops  of  a  20  per  cent  solution  of  sodium  carbonate  and  10  drops  of  a  20  per 
cent  solution  of  calcium  chloride.  Filter  off  the  resultant  precipitate  upon  a 
hardened  filter  paper  and  wash  it  with  water.  Remove  the  precipitate  to  a  small 
porcelain  dish,  add  3  c.c.  of  an  acid-alcohol  mixture^  and  a  few  drops  of  a  dilute 
solution  of  sodium  nitrite  and  heat.  The  production  of  a  green  color  indicates 
the  presence  of  bile  pigments. 

(h)  Bonanno's  Reaction.- — Place  5-10  c.c.  of  diluted  bile  in  a  small  porcelain 
evaporating  dish  and  add  a  few  drops  of  Bonanno's  reagent.^  An  emerald-green 
color  will  develop. 

5.  Tests  for  Bile  Acids. — (a)  Sucrose-HoSO^  Test  (Pettenkofer). — To  5  c.c 
of  diluted  bile  in  a  test-tube  add  5  drops  of  a  5  per  cent  solution  of  sucrose.  Now 
run  about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully  down  the  side  of  the 
tube  and  note  the  red  ring  at  the  point  of  contact.  Upon  slightly  agitating  the 
contents  of  the  tube  the  whole  solution  gradually  assumes  a  reddish  color.  As 
the  tube  becomes  warm,  it  should  be  cooled  in  rurming  water  in  order  that  the 
temperature  of  the  solution  may  not  rise  above  7o°C. 

It  is  claimed  that  this  test  is  not  satisfactory  in  the  presence  of 
protein  and  chromogenic  substances  which  yield  interfering  colors 
with  sulphuric  acid. 

(b)  Furfural-H2S04  Test.— Mylius's  Modification  of  Pettenkofer's  Test.— 
To  approximately  5  c.c.  of  diluted  bile  in  a  test-tube  add  3  drops  of  a  very  dilute 
(i  :  1000)  aqueous  solution  of  furfural, 

HC— CH 

II      II 
HC     CCHO. 


O 

Now  run  about  23  c.c.  of  concentrated  sulphuric  acid  carefully  down  the  side  of 
the  tube  and  note  the  red  ring  as  above.  In  this  case,  also,  upon  shaking  the 
tube  the  whole  solution  is  colored  red.  Keep  the  temperature  of  the  solution  be- 
low 7o°C.  as  before. 

(c)  Foam  Test  (v.  Udransky). — ^To  5  c.c.  of  diluted  bile  in  a  test-tube  add  3-4 
drops  of  a  very  dilute  (i  :  1000)  aqueous  solution  of  furfural.  Place  the  thtunb 
over  the  top  of  the  tube  and  shake  the  tube  until  a  thick  foam  is  formed.  By 
means  of  a  small  pipette  add  2-3  drops  of  concentrated  sulphuric  acid  to  the  foam 
and  note  the  dark  pink  coloration  produced. 

^  Made  by  adding  5  c.c.  of  concentrated  hj'drochloric  acid  to  95  c.c.  of  96  percent 
alcohol. 

^11  Tommasi,  2,  No.  21. 

^  This  reagent  may  be  prepared  by  dissolving  2  grams  of  sodium  nitrite  in  100  c.c.  of 
concentrated  hydrochloric  acid. 


BILE 


209 


fd)  Surface  Tension  Test  (Hay). — This  test  is  based  upon  the  principle  that 
bile  acids  have  the  property  of  reducing  the  surface  tension  of  fluids  in  which  they 
are  contained.  The  test  is  performed  as  follows:  Cool  about  10  c.c.  of  diluted 
bile  in  a  test-tube  to  17 'C.  or  lower  and  sprinkle  a  Uttle  finely  pulverized  sulphur 
upon  the  surface  of  the  fluid.  The  presence  of  bile  acids  is  indicated  if  the 
sulphur  sinks  to  the  bottom  of  the  Uquid,  the  rapidity  with  which  the  sulphur  sinks 
depending  upon  the  quantity  of  bile  acids  present  in  the  mixture.  The  test  is  said 
to  react  with  bile  acids  when  they  are  present  in  the  ratio  of  i :  120,000. 

(e)  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few  drops  of 
diluted  bile  in  an  evaporating  dish  add  a  trace  of  a  dilute  sucrose  solution  and  one 
or  more  drops  of  dilute  sulphuric  acid.  Evaporate  on  a  water-bath  and  note  the 
development  of  a  violet  color  at  the  edge  of  the  evaporating  mixture.  Discontinue 
the  evaporation  as  soon  as  the  color  is  observed. 

(f)  Peptone  Test  (Oliver). — To  5  cc  of  diluted  bile  add  2-3  drops  of  acetic 
acid,  filtering  if  necessary.  Add  an  equal  volume  of  a  i  per  cent  solution  of 
Witte's  peptone  to  the  acid  solution.  A  precipitate  is  produced  which  is  insoluble 
in  excess  of  acetic  acid.     This  precipitate  is  a  compound  of  protein  and  bile  acids. 

6.  CrystaUization  of  Bile  Salts. — To  25  c.c.  of  undiluted  bile  in  an  evaporating 
dish  add  enough  animal  charcoal  to  form  a  paste  and  evaporate  to  dryness  on  a 
water-bath.  Remove  the  residue,  grind  it  in  a  mortar,  and  transfer  it  to  a  small 
fiask.  Add  about  50  c.c.  of  95  per  cent  alcohol  and  boil  on  a  water-bath  for  20 
minutes.  Filter,  and  add  ether  to  the  filtrate  until  there  is  a  shght  permanent 
cloudiness.  Cover  the  vessel  and  stand  it  away  until  crystallization  is  complete. 
Examine  the  crystals  under  the  microscope  and  compare  them  with  those  shown  in 
Fig-  55)  page  205.     Try  one  of  the  tests  for  bile  acids  upon  some  of  the  crystals. 

7.  Analysis  of  BiUary  Calcuh. — Grind  the  calculus  in  a  mortar  with  10  c.c. 
of  ether.     Filter. 


Filtrate  I. 

I 

Add  an  equal  volume  of  95  per  cent  alco- 
hol^ to  the  ether  extract,  allow  the  mix- 
ture to  evaporate  and  examine  for  choles- 
terol crystals  (Fig.  57,  page  210).  (For 
further  tests  see  Experiment  8,  p.  210.) 


Residue  I. 
(On  paper  and  in  mortar.) 

.1 

Treat  with  dilute  hydrochloric  acid  and 
filter. 


FUtrate  U. 

Test  for  calcium,  phosphates,  and  iro>i. 
Evaporate  remainder  of  filtrate  to  dry- 
ness in  porcelain  crucible  and  ignite. 
Dissolve  residue  in  dilute  hydrochloric 
acid  and  make  alkaline  with  ammonium 
hydroxide.     Blue  color  indicates  copper. 


Residue  n. 
(On  paper  and  in  mortar.) 
Wash  with  a  little  water.   Dry  the  filter  paper. 


Treat  with  ^  c.c.  chloroform  and  filter. 


Filtrate  m. 
Bilirubin. 
(Apply  test  for 
bile  pigments.) 


Residue  III. 
(On  paper  and  in  mortar.) 


Treat  with   5 
alcohol. 


of  hot 


Bilivcrdin. 
*  The  alcohol  is  added  because  of  the  fact  that  it  is  often  found  that  crystallization  from 
pure  ether  does  not  yield  typical  cholesterol  crystals. 

14 


2IO 


PHYSIOLOGICAL    CHEMISTRY 


8.  Tests   for    Cholesterol. 

(a)  Microscopical  Examination. — Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  shown  in  Fig.  57,  below. 

(b)  Sulphuric  Acid  Test  (Salkowski). — Dissolve  a  few  crystals  of  cholesterol 
in  a  Uttle  chloroform  and  add  an  equal  volxmie  of  concentrated  sulphuric  acid. 
A  play  of  colors  from  bluish-red  to  cherry-red  and  purple  is  noted  in  the  chloro- 
form while  the  acid  assmnes  a  marked  green  fluorescence. 

(c)  Acetic  Anhydride-H2S04  Test  (Liebermann-Burchard). — Dissolve  a 
few  crystals  of  cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  sulphuric  acid.  The 
solution  becomes  red,  then  blue,  and  finally  bluish-green  in  color.  This  reaction 
is  used  in  the  quantitative  determination  of  cholesterol  (see  Chapter  XVI). 

(d)  Iodine -sulphuric  Acid  Test. — Place  a  few  crystals  of  cholesterol  in  one  of 
the  depressions  of  a  test-tablet  and  treat  with  a  drop  of  concentrated  sulphuric  acid 
and  a  drop  of  a  very  dilute  solution  of  iodine.  A  play  of  colors  consisting  of  violet, 
blue,  green,  and  red  results. 


Fig.  57. — Cholesterol. 


(e)  Schiff's  Reaction. — To  a  little  cholesterol  in  an  evaporating  dish  add  a  few 
drops  of  a  reagent  made  by  adding  i  volume  of  10  per  cent  ferric  chloride  to  3  vol- 
umes of  concentrated  sulphuric  acid.  Evaporate  to  dryness  over  a  low  flame  and 
observe  the  reddish-violet  residue  which  changes  to  a  bluish-violet. 

9.  Preparation  of  Taurine. — To  300  c.c.  of  bile  in  a  casserole  add  100  c.c.  of 
hydrochloric  acid  and  heat  until  a  sticky  mass  (dyslysin)  is  formed.  This  point 
may  be  determined  by  drawing  out  a  thread-like  portion  of  the  mass  by  means  of  a 
glass  rod,  and  if  it  solidifies  immediately  and  assumes  a  brittle  character  we  may 
conclude  that  all  the  taurocholic  and  glycoholic  acid  has  been  decomposed.  Decant 
the  solution  and  concentrate  it  to  a  small  volume  on  the  water-bath.  Filter  the 
hot  solution  to  remove  sodium  chloride  and  other  substances  which  may  have  sepa- 
rated, and  evaporate  the  filtrate  to  dryness.  Dissolve  the  residue  in  5  per  cent 
hydrochloric  acid  and  precipitate  with  10  volumes  of  95  per  cent  alcohol.  Filter 
off  the  taurine  and  recrystallize  it  from  hot  water.  (Save  the  alcoholic  filtrate  for 
the  preparation  of  glycocoll,  p.  211.)  Make  the  following  tests  upon  the  taurine 
crystals. 


BILE 


211 


(a)  Examine  them  under  the  microscope  and  compare  with  Fig.  58. 

(b)  Heat  a  crj'stal  upon  platinum  foil.  The  taurine  at  first  melts,  then  turns 
brown,  and  finally  carbonizes  as  the  temperature  is  raised.  Note  the  suflfocating 
odor.     What  is  it? 


Fig.  58. — Taurine. 

(c)  Test  the  solubility  of  the  crystals  in  water  and  in  alcohol. 

(d)  Grind  up  a  crystal  with  four  times  its  volume  of  dry  sodium  carbonate  and 
fuse  on  platinum  foil.  Cool  the  residue,  transfer  it  to  a  test-tube,  and  dissolve  it  in 
water.  Add  a  little  dilute  sulphuric  acid  and  note  the  odor  of  hydrogen  sulphide. 
Hold  a  piece  of  filter  paper,  moistened  with  a  small  amount  of  lead  acetate,  over 
the  opening  of  the  test-tube  and  observe  the  formation  of  lead  sulphide. 


'Js>' 


■M» 
%>- 


Fig.  59. — Glycocoll. 

10.  Preparation  of  Glycocoll.— Concentrate  the  alcoholic  filtrate  from  the  last 
experiment  (g)  until  no  more  alcohol  remains.  The  glycocoll  is  present  here  in  the 
form  of  an  hydrochloride  and  may  be  liberated  from  this  combination  by  the  addi- 
tion of  freshly  precipitated  lead  hydroxide  or  by  lead  hydroxide  solution.  Remove 
the  lead  by  hydrogen  sulphide.  Filler  and  decolorize  the  filtrate  by  animal  char- 
coal. Filter  again,  concentrate  the  filtrate,  and  set  it  aside  for  crystallization. 
Glycocoll  separates  as  colorless  crystals  (Fig.  50). 


CHAPTER  XIII 
PUTREFACTION  PRODUCTS 

The  putrefactive  processes  in  the  intestine  are  the  result  of  the 
action  of  bacteria  upon  the  protein  material  present.  This  bacterial 
action  which  is  the  combined  effort  of  many  forms  of  micro-organisms 
is  confined  almost  exclusively  to  the  large  intestine.  Some  of  the  prod- 
ucts of  the  putrefaction  of  proteins  are  identical  with  those  formed 
in  tryptic  digestion,  although  the  decomposition  of  the  protein  material 
is  much  more  extensive  when  subjected  to  putrefaction.  Some  of  the 
more  important  of  the  putrefaction  products  are  the  following:  Indole, 
skatole,  paracresol,  phenol,  para-oxy phenylpro pionic  acid,  para-oxyphen- 
ylacetic  acid,  volatile  fatty  acids,  hydrogen  sulphide,  methane,  methyl 
mercaptan,  hydrogen,  and  carbon  dioxide,  besides  proteoses,  peptones, 
peptides,  ammonia,  and  amino-acids.  Basic  substances  such  as  choline, 
neurine,  putrescine  and  cadaverine  are  present  under  certain  conditions. 
Of  the  putrefaction  products  the  indole,  skatole,  phenol,  and  paracresol 
appear  in  part  in  the  urine  as  ethereal  sulphuric  acids,  whereas  the 
oxyacids  mentioned  pass  unchanged  into  the  urine.  The  potassium 
indoxyl  sulphate  (page  386)  content  of  the  urine  is  a  rough  indicator 
of  the  extent  of  the  putrefaction  within  the  intestine. 

The  portion  of  the  indole  which  is  excreted  in  the  urine  is  first  sub- 
jected to  a  series  of  changes  within  the  organism  and  is  subsequently 
eliminated  as  indican.     These  changes  may  be  represented  thus: 

/\         CH  /\         C(OH) 

I     II     +0-^      1     y 

CH  \/\/CH 


NH  NH 

Indole.  Indoxyl. 

/\         C(OH)                       /\  C(0-S03H) 

+H2SO4  —  I        I  II                             +H2O 


\/\/CH  \/\/CH 

NH  NH 

Indoxyl.  Indoxyl  sulphuric  acid. 

In  the  presence  of  potassium  salts  the  indoxyl  sulphuric  acid  is  then 
transformed  into  indoxyl  potassium  sulphate  (or  indican), 

^-X C(0-S03K), 

I       IJ 
\/\/CH 

NH 

and  eliminated  as  such  in  the  urine. 

212 


PUTREFACTION   PRODUCTS  213 

Indican  may  be  decomposed  by  treatment  with  concentrated  hydro- 
chloric acid  (see  tests  on  page  387)  into  sulphuric  acid  and  indoxyl. 
The  latter  body  may  then  be  oxidized  to  form  indigo-bluc  thus: 

/\         C(OH)  /\         COOC         /\v 

2    I  II  +20->|  I  I  ,     +2H2O 

\/CH  \/\xc.A.c\/\y 

NH  NH  NH 

Indoxyl.  Indigo-blue. 

This  same  reaction  may  also  occur  under  pathological  conditions 
within  the  organism,  thus  giving  rise  to  the  appearance  of  crystals  of 
indigo-blue  in  the  urine. 

Skatole  or  methyl  indole  possesses  the  following  structure: 

_C(CH3) 


\/\/CH 
NH 

In  common  with  indole  it  is  changed  within  the  organism  and  eliminated 
in  the  form  of  a  chromogenic  substance.  Skatole  is,  however,  of  less 
importance  as  a  putrefaction  product  than  indole  and  ordinarily  occurs 
in  much  smaller  amount.  The  tryptophane  group  of  the  protein  mole- 
cule yields  the  indole  and  skatole  formed  in  intestinal  putrefaction,  but 
the  reasons  for  the  transformation  of  the  major  portion  of  this  trypto- 
phane into  indole  and  the  minor  portion  into  skatole  are  not  well  under- 
stood.    Indole  is  more  toxic  than  skatole. 

Phenol  occurs  in  fairly  large  amount  in  certain  abnormal  conditions 
of  the  organism,  but  ordinarily  the  amount  is  very  small.  It  is  probably 
derived  from  the  tyrosine  group  of  the  protein  molecule.  Phenol  is 
conjugated  in  the  liver  to  form  phenyl  potassium  sulphate  and  appears 
in  the  urine  in  this  form  (Baumann,  and  Herter).  Para-cresol  occurs 
in  the  urine  as  cresyl  potassium  sulphate. 

Regarding  the  claim  of  Nencki  that  methyl  mercaptan  is  formed 
as  a  gas  during  intestinal  putrefaction  it  is  an  important  fact  that 
Herter^  was  unable  to  detect  the  mercaptan  in  fresh  feces.  He  was, 
therefore,  not  inclined  to  accept  the  theory  that  methyl  mercaptan  is 
formed  in  ordinary  intestinal  putrefaction  but  believed  that  it  may  be 
formed  in  exceptional  cases.  Hydrogen  sulphide  is,  however,  formed  in 
all  cases  of  intestinal  putrefaction. 

It  has  been  demonstrated  that  putrefaction  processes  in  the  human 
intestine  may  be  retarded  by  the  ingestion  of  a  carbohydrate  diet.'^  The 
putrefactive  organisms  are  facultative  organistfis  and  prefer  a  carbo- 

1  Herter:  "Bacterial  Infections  of  the  Digestive  Tract,  p.  227." 

^Kendall:  Jour.  Med.  Res.,  24,  411,  1911;  also  Pediatrics,  23,  No.  9,  1910. 


214  PHYSIOLOGICAL   CHEMISTRY 

hydrate  medium  if  it  is  available.  These  organisms  are  also  unable  to 
exert  their  maximum  activity  in  an  acid  medium  and  therefore  the 
acids  resulting  from  the  carbohydrate  fermentation  would  tend  to  lessen 
their  activity. 

It  has  been  shown  by  Kutscher  and  his  associates^  that  many  acids 
and  bases  formed  in  putrefaction  and  which  have  been  considered  as 
originating  alone  from  bacterial  action,  may  also  be  formed  in  certain 
phases  of  metabohsm  in  both  the  plant  and  animal  kingdoms.  These 
transformation  products  of  amino-acids  have  been  termed  "apor- 
rhegmas."  The  following  aporrhegmas  may  result  from  putrefaction 
processes: 

Aporrhegma  Atnino-acid  source 

Iminazolethylamine.  .^ \  Histidine. 

Iminazolpropionic  acid ) 

Ornithine ] 

Tetramethylendiamine [  Arginine. 

Aminovalerianic  acid J 

Pentamethylendiamine Lysine. 

Amino-butyric  acid Glutamic  acid. 

^^'^P?---., )  Aspartic  acid. 

Succinic  acid J       ^ 

Isovalerianic  acid Leucine. 

Phenylethylamine 1 

Phenylacetic  acid \  Phenylalanine. 

Phenylpropionic  acid J 

/>-Oxyphenylacetic  acid. \  Xyj-osine 

/>-0.xj^henylpropionic  acid J 

Indole 1 

Skatole. 1  Tryptophane. 

indolacetic  acid 

Indolpropionic  acid •.  J  . 

Experiments  on  Putrefaction  Products 

In  many  courses  in  physiological  chemistry  the  instructors  are  so 
limited  for  time  that  no  extended  study  of  the  products  of  putrefaction 
can  very  well  be  attempted.  Under  such  conditions  the  scheme  here 
submitted  may  be  used  profitably  in  the  way  of  demonstration.  Where 
the  number  of  students  is  not  too  great,  a  single  large  putrefaction  vaky 
be  started,  and,  after  the  initial  distillation,  both  the  resulting  distillate 
and  residue  may  be  distributed  to  the  members  of  the  class  for  individual 
manipulation. 

Preparation  of  Putrefaction  Mixture. — Place  a  weighed  mixture  of  coagulated 
egg  albumin  and  ground  lean  meat  in  a  flask  or  bottle  and  add  approximately 
2  liters  of  water  for  every  kilogram  of  protein  used.  Sterilize  the  vessel  and  con- 
tents, inoculate  with  the  colon  bacillus,  and  keep  at  40°C.  for  two  or  three  weeks. 
If  cultures  of  the  colon  bacillus  are  not  available,  add  6o  c.c.  of  a  cold  saturated 

1  Ackermann  and  Kutscher:  Zeil.  physiol.  Cliem.,  69,  265,  1910. 
Ackermann:  Ibid.,  273. 
Engeland  and  Kutscher:  Ibid.,  282. 


PUTREFACTION   PRODUCTS 


21:; 


solution  of  sodium  carbonate  for  every  liter  of  water  previously  added  and 
inoculate  with  some  putrescent  material  (pancreas  or  feces).'  Mix  the  putre- 
faction mixture  very  thoroughly  by  shaking  and  insert  a  cork  furnished  with  a 
glass  tube  to  which  is  attached  a  wash  bottle  containing  a  3  per  cent  solution  of 
mercuric  cyanide.'  This  device  is  for  the  purpose  of  collecting  the  methyl 
mercaptan,  a  gas  formed  during  the  process  of  putrefaction.  It  also  serves  to 
diminish  the  odor  arising  from  the  putrefying  material.  Place  the  putrefaction 
mixture  at  40°C.  for  two  or  three  weeks  and  at  the  end  of  that  time  make  a  sepa- 
ration of  the  products  of  putrefaction  according  to  the  following  directions  : 

Subject  the  mixture  to  distillation  until  the  distillate  and  residue  are  approxi- 
mately equal  in  volume. 

PART  I 
MANIPULATION  OF  THE  DISTILLATE 

Acidify  with  hydrochloric  acid  ahd  extract  with  ether. 


Ether  Extract  No.   i. 

Add  an  equal  \'olume  of  water,  make  alka- 
line with  potassium  hj-droxide,  and  shake 
thoroughly.  I 


Residue  No.   i. 
Allow  the  ether  to  volatilize, 
rate     and     detect     ammonium 
crystals  (Fig.  60,  page  216). 


Evapo- 
chloride 


Ether  Extract  No.  2. 
Evaporate     spontaneously.     Indole     and 
skatol   remain.     Try    proper  reactions   (see 
pages  218  and  219). 


Alkaline  Solution  No.   i. 
Acidify    with    hydrochloric    acid,    add 
sodium  carbonate,  and  extract  with  ether. 


Ether  Extract  No.  3. 
Evaporate.     Detect     phenol 
(paracresol).     See  page  219. 


Alkaline  Solution  No.  2. 

and     cresol  .\cidify    with    hydrochloric    acid, 

extract  with  ether. 


and 


Ether  Extract  No.  4. 

Evaporate.      Volatile  folly    acids    remain. 


Final  Residue. 

(  Discard.  1 


DETAILED   DIRECTIONS   FOR  MAKING   THE   SEPARATIONS 
INDICATED  IN  THE  SCHEME 


Preliminary  Ether  Extraction. — This  extraction  may  be  conveniently  conducted 
in  a  separatory  funnel.  Mix  the  fluids  for  extraction  in  the  ratio  of  two  volumes 
of  ether  to  three  volumes  of  the  distillate.  Shake  very  thoroughly  for  a  few  mo- 
ments, then  draw  off  the  extracted  fluid  and  add  a  new  portion  of  the  distillate. 
Repeat  the  process  until  the  entire  distillate  has  been  extracted.  Add  a  small 
amount  of  fresh  ether  at  each  extraction  lo  replace  that  dissolved  by  the  water  in 
the  preceding  extraction. 

'  Putrefying  pri)lein  may  be  prepared  1)\-  treating  10  grams  of  tinely  ground  lean  meat 
with  100  c.c.  of  water  and  2  c.c.  of  a  saturated  solution  of  sodium  carbonate  and  keeping 
the  mixture  at  40°C.  for  24  hours. 

*l$  -  Concentrated  sulphuric  acid  containing  a  small  amount  of  /,<.<;//;/  may  be  used  as  a 
substitute  for  mercuric  cyanide.  When  this  modilicalion  is  employed  it  is  necessary  to 
use  calcium  chloride  tubes  to  exclude  moisture  from  the  isatin  solution. 


2l6 


PHYSIOLOGICAL    CHEMISTRY 


■•  Residue  No.  i. — Unite  the  portions  of  the  distillate  extracted  as  above  and  allow 
the  ether  to  volatilize  spontaneously.  Evaporate  until  crystallization  begins. 
Examine  the  crystals  under  the  microscope.  Ammonium  chloride  predominates. 
Explain  its  presence. 

Ether  Extract  No.  i. — Add  equal  volume  of  water,  render  the  mixture  alkaline 
with  potassium  hydroxide,  and  shake  thoroughly  by  means  of  a  separatory  funnel 
as  before.  The  volatile  fatty  acids,  contained  among  the  putrefaction  products, 
would  be  dissolved  by  the  alkaline  solution  (No.  i)  whereas  any  indole  or  skatole 
would  remain  in  the  ethereal  solution  (No.  2). 

Alkaline  Solution  No.  i. — Acidify  with  hydrochloric  acid  and  add  sodium 
carbonate  solution  until  the  fluid  is  neutral  or  slightly  acid  from  the  presence  of 
carbonic  acid.  At  this  point  a  portion  of  the  solution,  after  being  heated  for  a  few 
moments,  should  possess  an  alkaline  reaction  on  cooling.  Extract  the  whole  mix- 
ture with  ether  in  the  usual  way,  using  care  in  the  manipulation  of  the  stop  cock  to 


Fig.  60. — Ammonium  Chloride. 


relieve  the  pressure  due  to  the  evolution  of  carbon  dioxide.  The  ether  (Ether 
Extract  No.  3)  removes  any  phenol  or  crcsol  which  may  be  present  while  the  volatile 
fatty  acids  will  remain  in  the  alkaline  solution  (No.  2)  as  alkali  salts. 

Ether  Extract  No.  2. — Drive  off  the  major  portion  of  the  ether  at  a  low  tempera- 
ture on  a  water-bath  and  allow  the  residue  to  evaporate  spontaneously.  Indole 
and  skatole  should  be  present  here.  Prove  the  presence  of  these  bodies.  For 
tests  for  indole  and  skatole  seepages  218  and  219. 

Alkaline  Solution  No.  2. — Make  strongly  acid  with  hydrochloric  acid  and  ex- 
tract with  a  small  amount  of  ether,  using  a  separatory  funnel.  As  carbon  dioxide  is 
liberated  here,  care  must  be  used  in  the  manipulation  of  the  stop  cock  of  the  funnel 
in  relieving  the  pressure  within  the  vessel.  The  volatile  fatty  acids  are  dissolved 
by  the  ether  (Ether  Extract  No.  4). 

Ether  Extract  No.  3. — Evaporate  this  ethereal  solution  on  a  water-bath.  The 
oily  residue  contains  phenol  and  cresol.  The  cresol  is  present  for  the  most  part  as 
paracresol.  Add  some  water  to  the  oUy  residue  and  heat  it  in  a  flask.  Cool  and 
prove  the  presence  of  phenol  and  cresol.     For  tests  for  these  bodies  see  page  219. 

Ether  Extract  No.  4. — Evaporate  on  a  water-bath.  The  volatile  fatty  acids 
remain  in  the  residue. 


PUTREFACTION   PRODUCTS 


217 


PART    II 

MANIPULATION  OF  THE  RESIDUE 

Evaporate,  filter,  and  extract  with  ether. 


Ether  Extract. 
Evaporate,   extract   the   residue   with 
warm  water,  and  filter. 


Aqueous  Solution. 
Evaporate    until    crj'Stals    begin    to 
form.     Stand    in    a    cold    place    until 
crystallization  is  complete.     Filter. 


Crystalline  Deposit. 
Consists  of  a  mixture    of 
leucine  and  tyrosine  crystals 
(Figs.  25,  28  and  139,  pages 
76,  80  and  463.) 


Filtrate  No.  2. 

Contains  oxyacids   and 
skatole-carbonic  acid. 


Residue. 

Contains  non-volatile 
fatty  acids. 


Filtrate  No.  i. 
Contains   proteose,   peptone, 
aromatic  acids,  and  tryptophane. 


DETAILED  DIRECTIONS  FOR  MAKING  THE 

SEPARATIONS  INDICATED  IN 

THE  SCHEME 


Preliminary  Ether  Extraction. — This  extraction  may  be  conducted  in  a  separatory 
funnel.  In  order  to  make  a  satisfactory  extraction  the  mixture  should  be  shaken 
thoroughly.  Separate  the  ethereal  solution  from  the  aqueous  portion  and  treat 
them  according  to  the  directions  given  on  page  215. 

Ether  Extract. — Evaporate  this  solution  on  a  safety  water-bath  until  the  ether 
has  been  entirely  removed.     Extract  the  residue  with  warm  water  and  filter. 

Aqueous  Solution. — Evaporate  this  solution  until  crystallization  begins.  Stand 
the  solution  in  a  cold  place  until  no  more  crystals  form.  This  crystalline  mass  con- 
sists of  impure  leucine  and  tyrosine.     Filter  off  the  crystals. 

Crystalline  Deposit. — Examine  the  crystals  under  the  microscope  and  compare 
them  with  those  reproduced  in  Figs.  25,  28,  and  139,  pages  76,  80,  and  463.  Do  the 
forms  of  the  crj'stals  of  leucine  and  tyrosine  resemble  those  previously  examined? 
Make  a  separation  of  the  leucine  and  tyrosine  and  apply  typical  tests  according  to 
directions  given  on  pages  85  and  86. 

Filtrate  No.  i. — Make  a  test  for  tryptophane  with  bromine  water  (see  page  189) , 
and  also  with  the  Hopkins-Cole  reagent  (see  page  98).  Use  the  remainder  of  the 
filtrate  for  the  separation  of  proteoses  and  peptones.  Make  the  separation  ac- 
cording to  the  directions  given  on  page  120. 

Filtrate  No.  2. — This  solution  contains  para-o.xyphenylacetic  acid,  para-oxy- 
phenylpropionic  acid  and  skatole-carbonic  acid.  Prove  the  presence  of  these 
bodies  by  appropriate  tests.  Tests  for  o.xyacids  and  skatole-carbonic  acid  are 
given  on  page  220. 


2l8  PHYSIOLOGICAL    CHEMISTRY 

TESTS  FOR  VARIOUS  PUTREFACTION  PRODUCTS 

Tests  for  Indole 

The  various  tests  for  indole  ond  skatole  may  be  carried  out  upon  an 
aqueous  solution  ot  these  products  or  upon  an  aqueous  solution  of 
the  residue  from  Ether  Extract  No.  2  (see  page  172).  A  distillate 
secured  by  distilling  a  putrefaction  mixture  first  in  alkaline  and  then 
in  acid  reaction  may  also  be  employed. 

1.  Herter's  j5-Naphthaquinone  Reaction. — (a)  To  a  dilute  aqueous  solution 
of  indole  (i :  500,000)  add  i  drop  of  a  2  per  cent  solution  of  /i-naphthaquinone- 
sodiiun-monosulphonate.  No  reaction  occurs.  Add  a  drop  of  a  10  per  cent 
solution  of  potassium  hydroxide  and  note  the  gradual  development  of  a  blue 
or  blue-green  color  which  fades  to  green  if  an  excess  of  the  alkali  is  added. 
Render  the  green  or  blue-green  solution  acid  and  note  the  appearance  of  a 
pink  color.     Heat  faciUtates  the  development  of  the  color  reaction. 

One  part  of  indole  in  one  million  parts  of  water  may  be  detected  by  means  of 
this  test  if  carefully  performed. 

(b)  If  the  alkah  be  added  to  a  more  concentrated  indole  solution  before  the 
the  introduction  of  the  naphthaquinone  the  course  of  the  reaction  is  different, 
particularly  if  the  indole  solution  is  somewhat  more  concentrated  than  that  men- 
tioned above  and  if  heat  is  used.  Under  these  conditions  the  blue  indole  com- 
pound ultimately  forms  as  fine  acicular  crystals  which  rise  to  the  surface. 

If  we  do  not  wait  for  the  production  of  the  crystalline  body  but  as  soon  as  the 
blue  color  forms,  shake  the  aqueous  solution  with  chloroform,  the  blue  color  dis- 
appears from  the  solution  and  the  chloroform  assumes  a  pinkish-red  hue. 
This  is  a  distinguishing  feature  of  the  indole  reaction  and  facilitates  the  differen- 
tiation of  indole  from  other  bodies  which  yield  a  similar  blue  color.  A  very  sat- 
isfactory method  for  the  quantitative  determination  of  indole  is  based  upon  the 
principle  underlying  this  test  (see  chapter  on  Feces). 

2.  Formaldehyde  Reaction  (Konto). — To  i  c.c.  of  the  material  under  exami- 
nation in  a  test-tube  add  3  drops  of  a  40  per  cent  solution  of  formaldehyde  and  i 
c.c.  of  concentrated  sulphuric  acid.  Now  agitate  the  mixture  and  note  the  appear- 
ance of  a  violet-red  color  if  a  trace  of  indole  is  present.  The  test  is  said  to  serve 
for  the  detection  of  indole  when  present  in  a  dilution  of  i :  700,000. 

Skatole  gives  a  yellow  or  brown  color  under  the  above  conditions. 

3.  Cholera-red  Reaction. — To  a  little  of  the  material  under  examination  in  a 
test-tube  add  one-tenth  its  voliune  of  a  0.02  per  cent  solution  of  potassium  nitrite 
and  mix  thoroughly.  Carefully  run  concentrated  sulphuric  acid  down  the  side 
of  the  tube  so  that  it  forms  a  layer  at  the  bottom.  Note  the  purple  color.  Neu- 
tralize with  potassium  hydroxide  and  observe  the  production  of  a  bluish-green 
color. 

4.  Nitroprusside  Reaction  (Legal). — To  a  small  amount  of  the  material  under 
examination  in  a  test-tube  add  a  few  drops  of  a  freshly  prepared  solution  of  sodium 
nitroprusside,  Na2Fe(CN) 5NO+  2H2O.  Render  alkaline  with  potassium  hydroxide 
and  note  the  production  of  a  violet  color.  If  the  solution  is  now  acidified  with 
glacial  acetic  acid  the  violet  is  transformed  into  a  blue. 


PUTREFACTION   PRODUCTS  2ig 

5.  Pine  Wood  Test. — Moisten  a  pine  splinter  with  concentrated  hydrochloric 
acid  and  insert  it  into  the  material  under  examination.  The  wood  assumes  a 
cherr>--red  color. 

6.  Nitroso-indole  Nitrate  Test. — .\cidify  some  of  the  material  under  examina- 
tion with  nitric  acid,  add  a  few  drops  of  a  potassium  nitrite  solution  and  note  the 
production  of  a  red  precipitate  of  nitroso-indole  nitrate.  If  the  residue  contains 
but  little  indole  simply  a  red  coloration  will  result.  Compare  this  result  with  the 
result  of  the  similar  test  on  skatole. 


Tests  for  Skatole 

1.  Herter's  Para-dimethylaminobenzaldehyde  Reaction.' — To  5  c.c.  of  the 
distillate  or  aqueous  solution  under  examination  add  i  c.c.  of  an  acid  solution  of 
para-dimethylaminobenzaldehyde-  and  heat  the  mixture  to  boiling.  A  purpUsh- 
blue  coloration  is  produced  which  may  be  intensified  through  the  addition  of  a 
few  drops  of  concentrated  hydrochloric  acid.  If  the  solution  be  cooled  under 
running  water  it  loses  its  purpUsh  tinge  of  color  and  becomes  a  definite  blue. 
The  solution  at  this  point  may  be  somewhat  opalescent  through  the  separation  of 
uncombined  para-dimethylaminobenzaldehyde.  Care  should  be  taken  not  to 
add  an  excess  of  hydrochloric  acid  inasmuch  as  the  end-reaction  has  a  tendency 
to  fade  under  the  influence  of  a  high  acidity. 

A  rough  idea  regarding  the  actual  quantity  of  skatole  in  a  mixture  may  be 
obtained  by  extracting  this  blue  solution  with  chloroform  and  subsequentiy 
comparing  this  chloroform  solution,  by  means  of  a  colorimeter  Duboscq  ,  ^^"ith 
the  maximal  reaction,  obtained  with  a  skatole  solution  of  known  strength. 

2.  Color  Reaction  with  Hydrochloric  Acid.— Acidify  some  of  the  residue  with 
concentrated  hydrochloric  acid.     Note  the  production  of  a  \'iolet  color. 

3.  Acidify  some  of  the  residue  with  nitric  acid  and  add  a  few  drops  of  a  potas- 
sium nitrite  solution.  Xote  the  white  turbidity.  Compare  this  result  with  the 
result  of  the  similar  test  on  indole. 


Tests  for  Phenol  and  Cresole 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  reagent.  A  red 
color  results.  Compare  this  test  with  the  similar  one  under  Tyrosine  see  page 
86). 

2.  Ferric  Chloride  Test. — Add  a  few  drops  of  neutral  ferric  chloride  solution 
to  a  Uttle  of  the  material  under  examination.     A  dirty  bluish-gray  color  is  formed. 

3.  Formation  of  Bromine  Compounds. — Add  some  bromine  water  to  a  little 
of  the  fluid  under  examination.  Note  the  crystalline  precipitate  of  tribrom- 
phenol and  tribromcresol.     The  reaction  for  phenol  is  as  follows: 

CeHoOH+sBro^CeH.BraOH-l-sHBr. 

Phenol.  Tribromphenol. 

'.Herter:  Bacterial  Injections  of  the  Digestive  Tract.  1907,  p.  141. 

*  Made  by  dissolving  5  grams  of  para-dimethylaminobenzaldehyde  in  100  c.c.  of  10 
per  cent  sulphuric  acid. 

'  If  the  color  does  not  appear  add  more  of  the  aldehyde  solution. 


2  20  PHYSIOLOGICAL    CHEMISTRY 

4.  Nitric  Acid  Test. — ^Add  some  nitric  acid  to  some  of  the  material  under 
examination.  Heat  and  note  a  yellow  color  due  to  the  production  of  picric  acid 
(trinitrophenol)  from  phenol.     This  is  the  reaction : 

C6H50H+3HN03^C6H2(N02)30H+3H20. 

Phenol.  Picric  acid. 

Tests  for  Oxyacids 

1.  Color  Test. — Test  a  little  of  the  solution  with  Millon's  reagent.  A  red  color 
results. 

2.  Bromine  Water  Test.^ — Add  a  few  drops  of  bromine  water  to  some  of  the 
filtrate.     A  turbidity  or  precipitate  is  observed. 

Test  for  Skatole -carbonic  Acid 

Ferric  Chloride  Test. — Acidify  some  of  the  filtrate  with  hydrochloric  acid,  add 
a  few  drops  of  ferric  chloride  solution,  and  heat.  Compare  the  end-reaction  with 
that  given  by  phenol. 


CHAPTER  XIV 
FECES 

The  feces  are  the  residual  mass  of  material  remaining  in  the  intes- 
tine after  the  full  and  complete  exercise  of  the  digestive  and  absorptive 
functions  and  are  ultimately  expelled  from  the  body  through  the  rectum. 

They  may  be  said  to  be  composed  of  the  following  substances: 

1.  Food  residues:  (a)  those  portions  of  the  food  which  have  escaped 
absorption,  and  (b)  that  part  of  the  diet  either  not  digested  or  incapable 
of  absorption. 

2.  The  remains  of  the  intestinal  and  digestive  secretions  not 
destroyed  or  reabsorbed. 

3.  Substances  excreted  into  the  intestinal  tract,  notably  salts  of 
calcium,  iron,  and  other  metals. 

4.  The  bacterial  flora  of  the  intestinal  tract. 

5.  Cellular  elements  to  which  may  be  added,  under  pathological 
conditions,  blood,  pus,  mucus,  serum,  and  parasites. 

6.  Abnormally:  enteroliths,  gall  stones,  and  pancreatic  calculi. 
The  amount  of  the  fecal  discharge  varies  with  the  individual  and  the 

diet.  Upon  an  ordinary  mixed  diet  various  authorities  claim  that  the 
daily  excretion  by  an  adult  male  will  aggregate  1 10-170  grams  with  a 
soUd  content  ranging  between  25  and  45  grams;  the  fecal  discharge  of 
such  an  individual  upon  a  vegetable  diet  will.be  much  greater  and  may 
even  be  as  great  as  350  grams  and  possess  a  solid  content  of  75  grams. 
In  the  author's  own  experience  the  average  daily  output  of  moist  feces, 
calculated  on  the  basis  of  data  secured  from  the  examination  of  over 
1000  stools,  was  about  100  grams.  The  variation  in  the  normal  daily 
output  being  so  great  renders  this  factor  of  very  little  value  for  diag- 
nostic purposes,  except  where  the  composition  of  the  diet  is  accurately 
known.  Lesions  of  the  digestive  tract,  a  defective  absorptive  function, 
or  increased  peristalsis  as  well  as  an  admixture  of  mucus,  pus,  blood, 
and  pathological  products  of  the  intestinal  wall  may  cause  the  total 
amount  of  excrement  to  be  markedly  increased.  An  idea  of  the  varia- 
tion of  the  percentage  of  dry  matter  in  the  feces  evacuated  after  the 
ingestion  of  different  diets  may  be  gathered  from  a  consideration  of  the 
following  table. ^ 

'Schmidt  &  Strasburger:  "Die  Fazes  des  Menschen",  Berlin  iqi5 

221 


222 


PHYSIOLOGICAL   CHEMISTRY 


INFLUENCE  OF  DIET  ON  FECAL  DRY  MATTER 


Diet 


Dry  Matter  Percent. 


Nursing  infant . 
Milk  ' 

[  Adult. 

Meat 

Bread 

Potatoes. . .  . 
Cabbage .... 
Mixed  Diet. . 


15.0 

28.0 
29.0 
25.0 
15-0 
4-4 
26.0 


The  fecal  pigment  of  the  normal  adult  is  hydrobilirubin  This, 
pigment  originates  from  the  bilirubin  which  is  secreted  into  the  intes- 
tine in  the  bile,  the  transformation  from  bilirubin  to  hydrobilirubin 
being  brought  about  through  the  activity  of  certain  bacteria.  Hydro- 
bilirubin is  sometimes  called  stercobilin 
and  bears  a  close  resemblance  to  urobilin 
or  may  even  be  identical  with  that  pig- 
ment. Neither  bilirubin  nor  biliverdin 
occurs  normally  in  the  fecal  discharge  of 
adults,  although  the  former  may  be  de- 
tected in  the  excrement  of  nursing  in- 
fants. If  these  pigments  are  found  in 
the  feces  of  adults,  they  indicate  an 
abnormally  rapid  transit  through  the 
large  bowel  thus  preventing  their  trans- 
formation into  hydrobilirubin.  Fre- 
quently, in  some  way  as  yet  unknown, 
probably  through  the  agency  of  certain  bacterial  processes,  color- 
less hydrobilirubinogen  (leucohydrobilirubin)  is  formed  which  after 
the  passage  of  the  movement  and  exposure  to  air  is  reconverted 
into  hydrobilirubin.  This  may  explain  in  some  cases  the  darken- 
ing of  the  stool  when  exposed  to  the  air.  The  most  important 
factor  in  determining  the  color  of  the  fecal  discharge  is  the  diet.  A 
mixed  diet,  for  instance,  produces  stools  which  vary  in  color  from  light 
to  dark  brown,  an  exclusive  meat  diet  gives  rise  to  a  brownish-black 
stool,  whereas  the  stool  resulting  from  a  milk  diet  is  invariably  light 
colored.  Certain  pigmented  foods,  such  as  the  chlorophyllic  vegetables 
and  various  varieties  of  berries,  each  afford  stools  having  a  characteristic 
color.  Certain  drugs  act  in  a  similar  way  to  color  the  fecal  discharge. 
This  is  well  illustrated  by  the  occurrence  of  green  stools  following  the  use 
of  calomel,  of  black  stools  after  bismuth  ingestion,  and  of  yellow  stools 
following  the  administration  of  rhubarb,  senna  or  santonin.     The  green 


Fig.     61. — Hematoidin     Crys- 

T.A.LS     FROM     ACHOLIC     StOOLS.       {v. 

Jaksch.) 

Color  of  crystals  same  as  the  color 
of  those  in  Fig.  56,  page  205. 


FECES  223 

color  of  the  calomel  stool  is  generally  believed  to  be  due  to  biliverdin. 
V.  Jaksch,  however,  claims  to  have  proven  this  view  to  be  incorrect 
since  he  was  able  to  detect  hydrobilirubin  (or  urobilin)  but  no  biliverdin 
in  stools  after  the  administration  of  calomel.  The  bismuth  stool  was  at 
one  time  thought  to  derive  its  color  from  the  black  sulphide  which  is 
formed  from  the  subnitrate  of  bismuth.  We  now  know^  that  the  color 
is  due  to  the  reduction  of  the  bismuth  compound  (subnitrate)  to  bismuth 
suboxide.  In  cases  of  biliary  obstruction  the  grayish-white  acholic 
stool  is  formed. 

Under  normal  conditions  the  odor  of  feces  is  due  to  skatole  and 
indole,  two  bodies  formed  in  the  course  of  putrefactive  processes  occur- 
ring within  the  intestine  (see  page  212).  Such  bodies  as  methane, 
methyl  mercaptan,  and  hydrogen  sulphide  may  also  add  to  the  disagree- 
able character  of  the  odor.  The  intensity  of  the  odor  depends  to  a 
large  degree  upon  the  character  of  the  diet,  being  very  marked  in  stools 
from  a  meat  diet,  much  less  marked  in  stools  from  a  vegetable  diet,  and 
frequently  hardly  detectable  in  stools  from  a  milk  diet.  Thus  the  stool 
of  the  infant  is  ordinarily  nearly  odorless  and  any  decided  odor  may 
generally  be  readily  traced  to  some  pathological  source. 

A  neutral  reaction  ordinarily  predominates  in  normal  stools,  although 
slightly  alkaline  or  even  acid  stools  are  met  with.  The  acid  reaction  is 
encountered  much  less  frequently  than  the  alkaline,  and  then  commonly 
only  following  a  vegetable  diet. 

Experiments  in  which  the  actual  hydrogen  ion  concentration  of  the 
feces  was  determined  indicate  that  the  reaction  of  the  excreta  is  uni- 
formly slightly  alkaline.-  Pronounced  dietary  changes,  e.g.,  low  protein 
diet,  high  protein  diet,  fasting,  water  drinking  with  meals,  produce  at 
most  only  minor  changes  in  the  reaction  of  the  feces. 

The  form  and  consistency  of  the  stool  is  dependent,  in  large  measure, 
upon  the  nature  of  the  diet.  Under  normal  conditions  the  consistency 
may  vary  from  a  thin,  pasty  discharge  to  a  firmly  formed  stool.  Stools 
which  are  exceedingly  thin- and  watery  ordinarily  have  a  pathological 
significance.  In  general  the  feces  of  the  carnivorous  animals  is  of  a 
firmer  consistency  than  that  of  the  herbivora. 

The  continued  ingestion  of  a  diet  which  is  very  thoroughly  digested 
and  absorbed  is  frequently  accompanied  by  the  formation  of  dry.  hard 
fecal  masses  (scybala).  Constipation  generally  results,  due  to  the  small 
bulk  of  the  feces  and  its  lack  of  moisture.  At  present  the  formation  of 
scybala  is  considered  pathological,  as  an  expression  of  spastic  constipa- 
tion.    To  counteract  this  tendency  toward  constipation  the  ingestion 

'  Quincke:  Miinch.  med.  Woch.,  p.  854,  1896. 

*  Howe  and  Hawk:  Jour.  Biol.  Chem.,  11,  129,  191  2. 


2  24  PHYSIOLOGICAL   CHEMISTRY 

of  agar-agar'^  has  been  suggested."  This  agar  is  relatively  indigestible 
and  readily  absorbs  water  (about  i6  times  its  weight),  thus  forming  a 
bulky  fecal  mass  which  is  sufficiently  soft  to  permit  of  easy  evacuation. 
The  function  of  agar  is  not  limited  to  its  use  in  connection  with  consti- 
pation ;  it  may  serve  in  other  capacities  as  an  aid  to  intestinal  therapeu- 
tics by  serving  as  a  vehicle  for  certain  drugs. ^ 

It  is  frequently  desirable  for  clinical  or  experimental  purposes  to 
make  an  examination  of  the  fecal  output  which  constitutes  the  residual 
mass  from  a  certain  definite  diet.  Under  such  conditions,  it  is  custom- 
ary to  cause  the  person  under  observation  to  ingest  some  substance,  at 
the  beginning  and  end  of  the  period  in  question,  which  shall  sufficiently 
differ  in  color  and  consistency  from  the  surrounding  feces  as  to  render 
comparatively  easy  the  differentiation  of  the  feces  of  that  period  from 
the  feces  of  the  immediately  preceding  and  succeeding  periods.  One 
of  the  most  satisfactory  methods  of  making  this  "separation"  is  by 
means  of  the  ingestion  of  a  gelatin  capsule  containing  about  0.2  gram  of 
powdered  charcoal  at  the  beginning  and  end  of  the  period  under  observa- 
tion. This  procedure  causes  the  appearance  of  two  black  zones  of  char- 
coal in  the  fecal  mass  and  thus  renders  comparatively  simple  the 
differentiation  of  the  feces  of  the  intermediate  period.  Carmine  (0.3 
gram)  may  be  used  in  a  similar  manner  and  forms  two  dark  red  zones. 
Some  similar  method  for  the  "separation  of  feces"  is  universally 
practised  in  connection  with  the  scientifically  accurate  type  of  nutrition 
or  metabolism  experiment  which  embraces  the  collection  of  useful  data 
regarding  the  income  and  outgo  of  nitrogen  and  other  elements. 

Among  the  macroscopical  constituents  of  the  feces  may  be  men- 
tioned the  following:  Intestinal  parasites,  undigested  food  particles, 
gall  stones,  pathological  products  of  the  intestinal  wall,  enteroliths, 
intestinal  sand,  and  objects  which  have  been  accidentally  swallowed. 

The  fecal  constituents  which  at  various  times  and  under  different 
conditions  may  be  detected  by  the  use  of  the  microscope  are  as  follows : 
Constituents  derived  from  the  food,  such  as  muscle  fibers,  connective- 
tissue  shreds,  starch  granules,  and  fat;  form  elements  derived  from 
the  intestinal  tract,  such  as  epithelium,  erythrocytes,  and  leucocytes; 
mucus;  pus  corpuscles;  parasites  and  bacteria.  In  addition  to  the  con- 
stituents named  the  following  crystalline  deposits  may  be  detected: 
cholesterol,  ko  pro  sterol,  soaps,  fatty  acid,  fat,  hematoidin,  '^triple  phos- 

^  Agar-agar  is  a  product  prepared  from  certain  types  of  Asiatic  sea-weed.  It  is  a  carbo- 
hydrate and  is  classified  as  a  galaclan  in  the  polysaccharide  group. 

2  Mendel:  Zent.f.  ges.  Physiol,  u.  Path,  des  Stofw.,  No.  17,  p.  i,  1908;  Schmidt:  Miinch. 
med.  Woch.,  52,  1970,  1905. 

^  Einhorn:  Berl.  klin.  Woch.,  49,  113,  1912. 


FECES  225 

phate,"  Charcot-Leyden  crystals,  and  the  oxalate,  carbonate,  phosphate, 
sulphate,  and  lactate  of  calcium.     (See  Figs.  64  to  69,  pp.  230  and  231.) 

The  koprosterol  of  the  feces  is  similar  to  cholesterol,  and  may  be 
formed  by  the  reduction  of  the  latter.  It  responds  to  cholesterol  color 
tests  and  has  the  same  solubility,  but  possesses  a  lower  melting-point 
and  crystallizes  in  fine  needles  instead  of  plates  such  as  cholesterol 
forms. 

The  detection  of  minute  quantities  of  blood  in  the  feces  ("occult 
blood")  has  recently  become  a  recognized  aid  to  a  correct  diagnosis  of 
certain  disorders.  In  these  instances  the  hemorrhage  is  ordinarily  so 
slight  that  the  identification  by  means  of  macroscopical  characteristics 
as  well  as  the  microscopical  identification  through  the  detection  of  ery- 
throcytes are  both  unsatisfactory  in  their  results.  Of  the  tests  given 
for  the  detection  of  "occult  blood"  the  benzidine  reaction  and  the 
ortho-tolidin  and  hematein  tests  (page  233)  are  probably  the  most 
satisfactory.  Since  "occult  blood"  occurs  with  considerable  regularity 
and  frequency  in  gastrointestinal  cancer  and  in  gastric  and  duodenal 
ulcer,  its  detection  in  the  feces  is  of  especial  value  as 
an  aid  to  a  correct  diagnosis  of  these  disorders.  Cer- 
tain precautions  are  essential,  such  as  the  establish- 
ment of  a  meat-free  diet  over  a  period  of  time  before 
the  specimen  is  collected.  (Feces  from  a  meat  diet 
will  give  an  occult  blood  reaction  with  some  of  the 

most  delicate  tests.)     Bleeding  from  the  bowel  such  Fig.  62.— Charcot- 
,  1     •  1  11         ^1  1     •    .  r    Leydex  Crystals. 

as  IS  seen  m  hemorrhoids,  as  well  as  the  admixture  of 

menstrual  blood,  is  to  be  considered  in  the  interpretation  of  the  result. 

It  has  been  quite  clearly  shown  that  the  intestine  of  the  newly  born 
is  sterile.  However,  this  condition  is  quickly  altered  and  bacteria  may 
be  present  in  the  feces  before  or  after  the  first  ingestion  of  food.  There 
are  three  possible  means  of  infecting  the  intestine,  i.e.,  by  way  of  the 
mouth  or  anus  or  through  the  blood.  The  infection  by  means  of  the 
blood  seldom  occurs  except  under  pathological  conditions,  thus  limit- 
ing the  general  infection  to  the  mouth  and  anus. 

In  infants  with  pronounced  constipation  two-thirds  of  the  dry  sub- 
stance of  the  stools  has  been  found  to  consist  of  bacteria.  In  the  stools 
of  normal  adults  probably  about  one-third  of  the  dry  substance  is 
bacteria.^  The  average  excretion  of  dry  bacteria  in  24  hours  for  an 
adult  is  about  8  grams.  The  output  of  fecal  bacteria  has  been  found 
to  undergo  a  decrease  under  the  influence  of  water  drinking  with  meals. - 

'  Schittenhelm  and  Tollens  found  bacteria  to  comprise  42  per  cent  of  the  dry  matter. 
This  value  is,  however,  undoubtedly  too  high. 

-  Mattill  and  Hawk:  Jour.  Am.  Chan.  Soc,  33,  1999,  1911;  Blatherwick  and  Hawk: 
Bioch.  Bull.,  3,  28,  1913.^ 


2  26  PHYSIOLOGICAL   CHEMISTRY 

There  was  also  a  decrease  in  intestinal  putrefaction/  a  fact  which 
indicates  that  at  least  a  part  of  the  bacterial  deficit  was  made  up  of 
putrefactive  organisms.  In  some  cases  over  50  per  cent  of  the  total 
nitrogen  of  feces  has  been  shown  to  be  bacterial  nitrogen.'^ 

Various  enzymes  have  been  detected  in  the  feces.  The  first  one  so 
demonstrated  was  pancreatic  amylase.^  The  amylase  content  of  the 
feces  is  beheved  to  be  an  index  of  the  activity  of  the  pancreatic  function.^ 
The  excretion  of  this  enzyme  has  been  found  to  increase  under  the 
influence  of  water  drinking  with  meals. ^  Other  enzymes  which  have 
been  found  in  the  feces  under  various  conditions  are  tripsin,  rennin, 
maltase,  sucrase,  lactase,  nuclease  and  lipase.^  In  an  abnormally  rapid 
transit  of  food  through  the  intestinal  tract,  such  as  is  seen  in  certain 
diarrheas,  nearly  all  of  these  enzymes  may  be  detected. 

Some  of  the  more  important  organisms  met  with  in  the  feces  are  the 
following:'^  B.  coli,  B.  lactis  aerogenes,  Bad.  Welchii,  B.  bifidus,  and 
coccal  forms.  Of  these  the  first  three  types  mentioned  are  gas-forming 
organisms.  The  production  of  gas  by  the  fecal  flora  in  dextrose- 
bouillon  is  subject  to  great  variations  under  pathological  conditions; 
alterations  in  the  diet  of  normal  persons  will  also  cause  wide  fluctuations. 
Data  as  to  the  production  of  gas  are  of  considerable  importance  in  a 
diagnostic  way,  although  the  exact  cause  of  the  variations  is  not  yet 
established.  It  should  be  borne  in  mind  in  this  connection  that  gas 
volumes  are  frequently  variable  with  the  same  individual.  For  this 
reason  it  is  necessary  in  every  instance  to  follow  the  gas  production  for 
a  considerable  period  of  time  before  drawing  conclusions.^  While  the 
question  of  the  study  of  bacterial  flora  of  the  feces  is  a  question  beyond 
the  range  of  this  work,  mention  may  be  made  here  of  the  character  of 
the  organisms  observed  by  Gram  staining  of  the  stool  after  administra- 
tion of  different  types  of  diet.  It  has  been  shown  that  when  the  diet 
is  markedly  protein,  the  protein  type  of  flora  becomes  predominant  in 
the  stools.  Gram-stained  smears  show  a  fairly  equal  distribution  of 
Gram-negative  and  Gram-positive  organisms.  Among  the  latter  are 
largely  the  subtiloid  organisms  with  some  of  the  Bad.  Welchii,  together 
with  a  moderate  number  of  diplococci  and  coccoid  forms.  Most  of  the 
Gram-negative  organisms  resemble  the    B.  coli.     When  the    diet  is 

1  Hattrem  and  Hawk:  Arch.  Int.  Med.,  7,  610,  191 1;  Blatherwick,  Sherwin  and  Hawk: 
loc.  cit. 

^MacNeal,  Latzer  and  Kerr:  Jour.  Inf.  Dis.,  6,  123,  1909;  Mattill  and  Hawk:  Jour. 
Exp.  Med.,  14,  433,  1911;  Blatherwick  and  Hawk:  Biochem.  Bull.,  3,  28,  1913. 

^  Wegscheider:  Inaug.  Diss.,  Strassburg,  1875. 

*  Wohlgemuth:  Berl.  klin.  Woch.,  47,  3,  92,  1910. 

*  Hawk:  Arch.  Int.  Med.,  8,  382,  191 1. 
^Ury:  Biochem.  Zeit.,  23,  152,  1909. 

'  Herter  and  Kendall:  Journal  of  Biological  Chemistry,  5,  283,  1908. 

*  Herter  and  Kendall:  loc.  cit. 


FECES  227 

carbohydrate  the  field  is  strongly  Gram  positive  and  has  a  more  homo- 
geneous appearance.  The  bacteria  seen  consist  chiefly  of  long  slender 
Gram-positive  rods  belonging  to  the  B.  acidophilus  and  B.  bifidus 
groups.^ 

The  nitrogen  present  in  the  feces  consists  principally  of  bacteria, 
unahsorbed  intestinal  secretions,  epithelial  cells,  mucus  material  and  food 
residues.  In  the  early  days  of  nutrition  study  the  fecal  nitrogen  was 
believed  to  consist  principally  of  food  residues.  We  now  know  that 
such  residues  ordinarily  make  up  but  a  small  part  of  the  nitrogen  quota 
of  the  stools  of  normal  individuals  who  exercise  normal  mastication.^ 
When  meat  has  been  ''bolted,"  however,  from  3^  gram  to  16  grams  of 
macroscopical  meat  residues  has  been  found  in  a  single  stool. ^  The 
phrase  ''metabolic  product  nitrogen"  is  frequently  used  as  a  designa- 
tion for  all  fecal  nitrogen  except  that  present  as  food  residues  and 
bacteria.  Bacteria  cannot  logically  be  classed  under  "metaboHc" 
nitrogen  since  they  doubtless  develop  at  the  expense  of  food  nitrogen 
as  well  as  at  the  expense  of  that  in  the  form  of  intestinal  secretions. 
In  the  accurate  study  of  "protein  utilization"^  a  correction  should  be 
made  for  "metabolic  nitrogen."  Data  regarding  the  output  of  meta- 
bolic nitrogen  may  be  secured  by  determining  the  fecal  nitrogen  excre- 
tion on  a  diet  of  proper  energy  value  but  containing  no  nitrogen.^ 
Agar-agar  may  be  utilized  advantageously  in  connection  with  such  a 
nitrogen-free  diet. 

Feces  are  still  excreted  from  the  intestine  even  when  no  food  is 
ingested.  Carefully  conducted  fasting  experiments  have  demonstrated 
this.  A  dog  nourished  on  an  ordinary  diet  to  which  bone  ash  has  been 
added  will  excrete  a  grey  feces.  WTien  fasted  such  an  animal  will,  after 
a  few  days,  excrete  a  small  amount  of  a  greenish-brown  mass,  containing 
no  bone  ash.  These  are  fasting  feces.  It  is  of  a  pitch-like  consistency 
and  turns  black  on  contact  with  the  air.^  Adult  fasting  men  have  been 
found  to  excrete  7-8  grams  of  feces  per  day,  the  daily  nitrogen  value 
being  about  o.i  gram.^  Xo  separating  medium  such  as  charcoal  or 
carmine  (page  238)  should  be  used  in  differentiating  fasting  feces. 

In  recent  years  the  examination  of  feces  for  evidences  of  parasitism 
(detection  of  parasites  and  their  ova)  has  taken  on  an  added  importance. 
The  investigation  of  the  hookworm  has  been  particularly  developed. 

^  Cammidge:  The  Feces  of  Children  and  Adults,  1914,  p.  126. 

-  Kermauner:  Zeit.  fiir  Biol.,  35,  316,  1897. 

'  Foster  and  Hawk:  Jour.  Am.  Chem.  Soc,  37,  1347,  191 5. 

*  The  percentage  of  the  ingested  protein  which  is  absorbed  from  the  intestine.  This 
may  be  calculated  by  subtracting  the  metabolic  nitrogen  from  the  total  fecal  nitrogen  and 
dividing  this  value  by  the  food  nitrogen. 

'  Tsuboi:  Zeit.  jUr  Biol.,  35,  68,  1897;  Mendel  and  Fine:  Jour.  Biol.  Chem.,  11,  5,  191^. 

*  Howe  and  Hawk:  Jour.  Am.  Chem.  Soc,  n,  215,  1911. 
^  Howe,  Mattill  and  Hawk:  Ibid.,  33,  568,  191 1. 


'2-28  PHYSIOLOGICAL    CHEMISTRY 

(For  methods  and  discussion  see  Bulletin  135,  Bureau  of  Animal  Indus- 
try, U.  S.  Department  of  Agriculture,  191 1,  M.  C.  Hall.) 

For  diagnostic  purposes  the  macroscopical  and  microscopical  exami- 
nations of  the  feces  ordinarily  }deld  much  more  satisfactory  data  than 
are  secured  from  its  chemical  examination.  Possibly  with  the  excep- 
tion of  certain  examinations  for  occult  blood,  the  most  satisfactory  data 
for  diagnostic  purposes  are  secured  by  microscopical  examination. 
This  presupposes  a  knowledge  of  microscopical  technic  and  the  use  of 
certain  microchemical  tests,  by  which  much  information  can  be  ob- 
tained. The  principle  underhdng  this  examination  consists  in  the  study 
of  the  actual  changes  which  the  various  food-stuffs  have  undergone  dur- 
ing digestion.  A  knowledge  of  the  changes  which  occur  in  normal  diges- 
tion and  which  are  seen  in  normal  feces  enables  one  to  readily  detect 
pathological  variations.  One  diet  widely  used  for  this  purpose  is  the 
Schmidt  diet  which  is  given  below.  The  modification^  described  is 
better  adapted  to  American  conditions. 

The  Schmidt  intestinal  diet  is  as  follows: 

In  the  morning:  0.5  Hter  of  milk,  or  if  milk  does  not  agree  0.5  liter 
of  cocoa  (prepared  from  20  grams  of  cocoa  powder,  10  grams  sugar, 
400  grams  water,  and  100  grams  milk).     To  this  add  50  grams  zwiebach. 

In  the  forenoon:  0.5  Hter  oatmeal  gruel  (made  from  40  grams  oat- 
meal), 10  grams  butter,  100  grams  milk,  300  grams  water,  i  egg  strained. 

At  noon:  125  grams  of  chopped  beef  (raw  weight)  broiled  rare  with 
20  grams  of  butter,  so  that  the  interior  will  remain  raw.  To  this  add 
250  grams  potato  broth  (made  of  190  grams  mashed  potatoes,  100  grams 
milk,  10  grams  of  butter). 

In  the  afternoon:  as  in  the  morning. 

In  the  eventing:  as  in  the  forenoon. 

This  diet  necessitates  five  meals  a  day  especially  prepared  and  does 
not  follow  the  average  American  dietary.  In  simple  microscopical 
examinations  for  food  digestion,  the  following  diet  as  more  closely 
approximating  the  ordinary  dietary  regime  is  suggested.  Should 
chemical  determinations  for  fat  be  desired  all  fat  containing  foods  can 
be  ehminated  except  those  in  which  its  specific  content  is  known  and  a 
measured  amount  of  fat  given.  The  feces  can  then  be  separated  by 
means  of  carmine. 

Modified  Schmidt  Diet 
Breakfast: 

100  grams  Cream  of  wheat  or  oatmeal 
60  grams  toast 
20  grams  butter 
250  c.c.  milk. 

1  Used  by  Dr.  Rehfuss  at  Jefferson  Hospital. 


TECES 


229 


Luncheon: 

Rice  soup  (chicken  broth  with  rice) 
100  grams  green  vegetable  (asparagus) 
100  grams  mashed  potato 
60  grams  toast 
20  grams  butter 
250  c.c.  milk. 

4  o'clock: 

250  c.c.  of  milk. 

Dinner: 

150  grams  of  chopped  meat,  grilled  on  the  outside  and  rare  in  the  center 
100  grams  green  vegetable  (spinach) 
100  grams  mashed  potatoes 

60  grams  of  toast 

20  grams  of  butter 
250  c.c.  milk 
Stewed  fruit. 

Experiments  on  Feces 

I.  Macroscopical  Examination. — If  the  stool  is  watery  pour  it  into  a  shallow 
dish  and  examine  directly.  If  it  is  firm  or  pasty  it  should  be  treated  with  water 
and  carefully  stirred  before  the  examination  for  macroscopical  constituents  is 
attempted.  The  macroscopical  constituents  may  be  collected  very  satisfactorily 
by  means  of  a  double  layer  of  cheese  cloth. 

A  Boas  sieve  (Fig.  63)  may  also  be  used  to  collect  the  macroscopical 
constituents  of  feces.  This  sieve  is  constructed  of  two  easily  detachable 
hemispheres  which  are  held  together  by  means  of  a 
bayonet  catch.  In  using  the  apparatus  the  feces  is 
spread  out  upon  a  very  fine  sieve  contained  in  the 
lower  hemisphere  and  a  stream  of  water  is  allowed  to 
play  upon  it  through  the  medium  of  an  opening  in 
the  upper  hemisphere.  The  apparatus  is  provided 
with  an  orifice  in  the  upper  hemisphere  through 
w^hich  the  feces  may  be -stirred  by  means  of  a  glass 
rod  during  the  washing  process.  After  1 5-30  minutes 
washing  nothing  but  the  coarse  fecal  constituents 
remain  upon  the  sieve. 


Fig.  63. — Bo.\s 

SiF.VF. 


2.  Microscopical  Examination. — After  the  ingestion  of 
the  test  diet  (see  Schmidt  diet  above)  for  several  days,  a 
specimen  of  the  movement  is  collected.  Any  gross  abnor- 
malities are  recorded  in  the  form,  consistence,  and  char- 
acter of  the  stool  as  well  as  the  admixture  of  certain  pathological  elements 
such  as  pus,  blood,  mucus,  and  parasites.  The  movement  is  then  rubbed  out 
on  plates  and  the  presence  of  undigested  food-stuffs  sought  for.  Normally  the 
test  diet  is  almost  completely  digested  and  no  gross  undigested  material  is 
found.  Therefore  the  presence  of  these  macroscopic  rests  is  in  itself  evidence 
of  disturbed  digestion.  Clean  slides  and  cover-glasses  are  then  prepared 
and  a  small  representative  portion  of  the  movement  is  placed  on  each  of  three 
slides.     The  routine  clinical  method  of  examination  follows :     To  the  first  slide 


230  PHYSIOLOGICAL   CHEMISTRY 

is  added  a  drop  of  distilled  water  and  it  is  then  examined  with  low  and  high 
powers. 

Meat  fibers  are  readily  recognized  by  their  yellowish  hyaline  ap- 
pearance possibly  with  a  few  striee  still  visible  in  the  libers.     Should 


Fig.  64. — .4,  intact  undigested  meat  Fig.  65. — A,  neutral  fat;  B,  iatty  acid 

fibers;  B,  partially  digested  meat  fibers;  liberated  by  acetic  acid;  C,  soaps;  D,  fatty 

C,    almost    completely    digested    meat  acid  crystals, 
fibers. 


Fig.  66. — A,  elastic  tissue;  B,  white  Fig.  67. — .4 ,  cellulose  remains  of  vege- 

fibrous   tissue    (macroscopic);    C,    white  tables;  B,  empty   potato  cells;  C,   potato 

fibrous  tissue  (microscopic.)  cells   filled  with  starch,  and  stained  with 

iodine;  D.  hard  cells  found  in  pears;  E,  spiral 
and  woody  fibers  from  pith  of  vegetables; 
F,  vegetable  hairs. 

Figs.  64  to  67. — ^Iicroscopic.a.l  Constituents  of  Feces. 
meat   fibers   be   found   bound  together  by  connective   tissue  or  raw 
connective  tissue,  either  white  fibrous  or  yellow  elastic,  be  noted,  it 
indicates  a  disturbance  of  gastric  function  inasmuch  as  one  of  the  spe- 
cific functions  of  the  gastric  juice  is  to  dissolve  the  intercellular  tissue 


FECES 


231 


binding  together  the  fibers.  If  large  numbers  of  meat  fibers  are  found 
after  a  test  diet,  particularly  if  the  nuclei  are  still  intact  in  the  fibers, 
the  inference  of  poor  or  low  pancreatic  function  is  justifiable.  This 
is  true  if  it  can  be  demonstrated  that  the  food  has  been  sufficiently 
long  in  its  transit  through  the  intestinal  tract  to  permit  the  pancreatic 
enzymes  to  carry  on  their  work.  A  dilute  solution  of  methylene  blue 
will  readily  show  the  nuclei  if  present. 

The  second  slide  is  examined  for  fats  and  then  treated  with  acetic 
acid  and  heated  to  split  any  soaps  which  may  be  present  and  form 
fatty  acid. 

Fats  are  met  with  in  three  forms  (a)  neutral  fats  readily  demonstrated  by 
Sudan  III,  Scharlach  R  or  Osmic  acid;  (b)  fatty  acids  which  are  usually  found  in 
the  form  of  needle-like  crystals  soluble  in  ether,  alcohol,  and  solutions  of  sodium 


Fig.  69. — A,  Schmidt  test  bag  for  study 
of  pancreatic  function;  B,  nuclei  of  meat 
fibers  digested;  C,  nuclei  of  meat  fibers 
undigested;  D,  undigested  stained  thymus 
cells. 
Figs.  68  and  69.— Microscopical  Coxstituents  of  Feces. 


Fig.  68. — .4,  calcium  sulphate  crys- 
tals; B,  cholesterol  crystals;  C,  char- 
coal detritus;  D,  bismuth  sub-oxide 
crystals;  E,  calcum  oxalate  crystals. 


hydrate  (these  crystals  do  not  stain  with  Sudan  III  but  form  drops  on  being 
warmed) ;  (c)  soaps  are  usually  found  in  the  feces  either  as  amorphous  flakes  or 
scallop  shell-like  formations,  but  may  occasionally  occur  in  crystalline  form. 
The  calciiun  soaps  which  compose  the  bulk  of  the  soaps  in  the  feces  can  be  dis- 
tinguished from  the  potassium  and  sodiimi  compounds  because  of  their  insolu- 
bility in  hot  water,  alcohol,  and  ether.  On  heating  with  30  per  cent  acetic  acid, 
fatty  acids  are  set  free  in  drops  which  crystaUize  out  on  cooling. 

The  estimation  of  fats  is  a  rather  important  matter  and  the  trained 
observer  can  usually  detect  disturbances  in  fat  digestion.  Normally 
there  are  fats  present  in  the  movement,  but  abnormally  their  c}uantity 
is  relatively  increased  either  in  total  fat,  or  in  one  of  its  components. 


232  PHYSIOLOGICAL   CHEMISTRY 

While  it  is  true  that  bacterial  activity  plays  a  considerable  role  in  the 
digestion  of  fats,  a  marked  increase  in  fat  usually  indicates  pancreatic 
disease,  or  a  disturbance  in  pancreatic  function.  This  is,  of  course,  the 
case  only  when  the  amount  of  fat  ingested  is  not  in  excess  of  that  which 
can  be  readily  handled  under  normal  conditions.  In  cases  of  pure 
biliary  obstruction  without  pancreatic  involvement,  fat-splitting  takes 
place  in  a  normal  way,  but  the  fatty  acids  and  soaps  formed  are  not 
absorbed  owing  to  the  absence  of  bile.  Such  a  movement  is  full  of 
soaps  and  fatty  acid  crystals  which  on  treatment  with  acetic  acid  show 
a  marked  increase  in  total  fat  over  normal.  Failure  of  absorption 
owing  to  extensive  disease  of  the  intestinal  mucosa  can  produce  a  similar 
picture  but  will  usually  give  some  cytological  evidence  of  intestinal 
disease.  Pure  pancreatic  disease  gives  a  marked  increase  in  total  and 
neutral  fat  with  the  presence  of  bile. 

Undigested  starches  are  readily  recognized  by  their  blue  reaction  with  iodine. 
This  can  be  studied  on  the  third  sUde. 

This  phenomenon  is  the  least  frequent  among  the  different  forms  of 
pathological  digestion  and  usually  indicates  food  bolting,  an  excessive 
ingestion  of,  or  poor  preparation  of  carbohydrate  food,  or  an  infection 
of  the  bowel  with  so-called  "garungsdyspepsia"  rather  than  an  actual 
disturbance  of  pancreatic  function  inasmuch  as  theamylolytic  function 
of  the  pancreas  is  the  most  persistent  and  the  last  to  disappear. 

Disturbance  in  cellulose  digestion,  the  presence  of  blood,  leucocytes, 
mucus,  etc. ,  can  all  be  demonstrated  by  appropriate  technic  and  represent 
a  chapter  in  the  study  of  the  feces  ofgreat  diagnostic  importance,  but  one 
which  is  beyond  the  province  of  this  volume.  (For  further  discussion, 
see  page  228.  For  cuts  of  fecal  constituents  found  microscopically, 
see  pages  230  and  231.) 

3.  Reaction. — ^Thoroughly  mix  the  feces  and  apply  moist  red  and  blue  litmus 
papers  to  the  surface.  If  the  stool  is  hard  it  should  be  mixed  with  water  before 
the  reaction  is  taken.  Examine  the  stool  as  soon  after  defecation  as  is  convenient, 
since  the  reaction  may  change  very  rapidly.  The  reaction  of  the  normal  stools  of 
adult  man  is  ordinarily  neutral  or  faintly  alkaline  to  litmus,  but  seldom  acid. 
Infants'  stools  are  generally  acid  in  reaction.  Try  the  reaction  to  Congo  red 
paper.     Also  test  the  reaction  of  fecal  extract  to  phenolphthalein. 

4.  Starch. — If  any  imperfectly  cooked  starch-containing  food  has  been 
ingested  it  will  be  possible  to  detect  starch  granules  by  a  microscopical  examina- 
tion of  the  feces.  If  the  granules  are  not  detected  by  a  microscopical  examination, 
the  feces  should  be  placed  in  an  evaporating  dish  or  casserole  and  boiled  with 
water  for  a  few  minutes.  Filter  and  test  the  filtrate  by  the  iodine  test  in  the  usual 
way  (see  page  45). 

5.  Cholesterol,  Koprosterol  and  Fat. — Introduce  about  5  grams  of  moist 
feces  into  a  100  c.c.  glass-stoppered  cylinder.     Add  30  c.c.  of  distilled  water  and 


FECES  233 

25  c.c.  of  ether,  then  stopper  the  cylinder  and  shake  vigorously  for  five  minutes. 
Allow  to  separate,  pour  or  pipette  ofif  the  ethereal  solution.  FUter  and  remove  the 
ether  by  evaporation.  The  residue  contains  cholesterol  and  the  mixed  fats  of 
the  feces.  For  every  gram  of  fat  add  about  i  12  grams  of  soUd  potassium  hy- 
droxide and  25  c.c.  of  95  per  cent  alcohol  and  boil  in  a  flask  on  a  water-bath  for 
one-half  hour,  maintaining  the  volume  of  alcohol  constant.  This  alcohohc- 
potash  has  saponified  the  mixed  fats  and  we  now  have  a  mixture  of  soaps, 
cholesterol  and  koprosterol.  Add  sodium  chloride,  in  substance,  to  the  mixture 
and  extract  with  ether  to  dissolve  out  the  cholesterol  and  koprosterol.  Remove 
the  ether  by  evaporation  and  examine  the  residue  microscopically  for  cholesterol 
and  koprosterol  crystals.  Try  any  of  the  other  tests  for  cholesterol  as  given  on 
page  210. 

6.  Blood. — Undecomposed  blood  may  be  detected  macroscopically. 
If  uncertain,  look  for  erythrocytes  under  the  microscope,  and  spectro- 
scopically  for  the  spectrum  of  oxyhemoglobin  (see  Absorption  Spectra, 
Plate  1). 

In  case  the  blood  has  been  altered  or  is  present  in  minute  amount 
("occult  blood"),  and  cannot  be  detected  by  the  means  just  mentioned, 
the  following  tests  may  be  tried: 

(a)  Benzidine  Reaction. — Make  a  thin  fecal  suspension  using  about  5  c.c.  of 
distilled  water,  and  heat  it  to  boiUng  to  render  oxidizing  enzymes  inactive. 
To  2  c.c.  of  a  saturated  solution  of  benzidine  in  glacial  acetic  acid  add  3  c.c.  of 
3  per  cent  hydrogen  peroxide  and  2-3  drops  of  the  cooled  fecal  suspension. 
A  clear  blue  color  appears  within  one  to  two  minutes  in  the  presence  of  blood. 
If  the  mixture  is  not  shaken  a  ring  of  color  will  form  at  the  top.  Minute  traces  of 
blood  are  more  easily  detected  by  the  latter  procedure. 

Wagner^  has  simplified  the  benzidin  test  so  that  it  can  be  applied 
much  more  conveniently. 

Slide  Modification. — Take  up  a  little  of  the  solid  stool  on  a  match,  smear  it 
on  an  object  glass  and  pour  the  reagent  over  it.  It  turns  blue  if  there  is  blood 
present  and  there  is  no  misleading  green  tint  from  fluid.  Make  the  solution 
as  follows:  Add  a  knife-tip  of  benzidine  to  2  c.c.  of  glacial  acetic  acid,  and 
add  20  drops  of  a  3  per  cent  solution  of  hydrogen  peroxide. 

By  this  dry  technic  there  is  no  danger  of  soiling  the  fingers,  and  the 
test  is  more  sensitive  than  the  usual  "wet"  benzidine  test. ,  The  smear 
of  stool  is  either  blue  or  it  is  not  blue.  The  rapidity  of  the  color  change 
gives  some  idea  as  to  the  proportion  of  blood  in  the  stool ;  with  much 
blood  present  the  change  to  blue  is  instantaneous. 

(b)  Ortho-tolidin  Test  (Ruttan  and  Hardisty)^ — To  i  c.c.  of  a  4  per  cent 

^Wagner:  Zeulbl.  fiir  Chirurgie,  41,  No.  28,  1914. 

2  Ruttan  and  Hardisty:  Canadian  Medical  Ass'n  Journal,  Nov.,  1912,  also  Biochemical 
Bull.,  2,  225,  1913. 


234  PHYSIOLOGICAL   CHEMISTRY 

glacial  acetic  acid  solution  of  o-tolidin^  in  a  test-tube  add  i  c.c.  of  the  solution 
under  examination  and  i  c.c.  of  3  per  cent  hydrogen  peroxide.  In  the  presence 
of  blood  a  bluish  color  develops  (sometimes  rather  slowly)  and  persists  for 
some  time  (several  hours  in  some  instances). 

This  test  is  said  to  be  as  sensitive  for  the  detection  of  occult  blood  in 
feces  and  stomach  contents  as  is  the  benzidine  reaction.  It  is  also 
claimed  to  be  more  satisfactory  for  urine  than  any  other  blood  test. 
The  acetic  acid  solution  may  be  kept  for  one  month  with  no  reduction 
in  delicacy. 

(c)  Phenol phthalein  Test^ — Make  a  thin  fecal  suspension  using  about  5  c.c.  of 
distilled  water.  Heat  to  boiling,^  cool  and  add  2  c.c.  of  the  suspension  to  i  c.c.  of 
the  phenolphthalein  reagent^  and  a  few  drops  of  hydrogen  peroxide.  A  pink  or 
red  color  promptly  forms  in  the  presence  of  blood. 

Schirokauer*  makes  the  statement  that  a  mixture  of  alcohol  and  glacial  acetic 
acid  will  give  the  phenolphthalein  reaction  for  occult  blood.  The  action  of  an  oxi- 
dizing agent  will  make  this  reaction  more  distinct.  Von  Czylharz  and  Neustadl® 
find  that  a  solution  of  sodium  salicylate  added  to  a  blood-free  extract  of  feces  will 
give  a  very  deceptive  reaction,  while  feces  after  the  administration  of  sodium 
salicylate  by  mouth  gave  the  same  reaction.  The  same  was  true  of  acetyl  salicylic 
acid  and  other  similar  drugs.  Their  studies  in  clinical  cases  likewise  indicated  that 
the  phenolphthalein  test  was  unreliable. 

{d)  Hematein  Reaction  for  Occult  Blood. — Couturier"  advises  the  use  of 
hematein^  in  testing  for  occult  blood.  It  is  only  slightly  soluble  in  water, 
but  gives  a  pronounced  red  color.  In  contact  with  sodium  hydroxide 
this  red  solution  turns  a  deep  violet  blue,  giving  an  insoluble  compound 
of  hematein  and  sodium.  This  compound,  exposed  to  the  air  oxidizes 
after  several  days,  and  gives  brownish  or  yellowish  compounds,  depend- 
ing on  dilution.  This  change  is  only  hastened  a  little  by  the  addition 
of  hydrogen  peroxide,  but  if  a  trace  of  blood  is  added  to  the  hydrogen 
peroxide,  it  takes  place  almost  instantly.  To  avoid  oxidation,  the  hem- 
atein sodium  mixture  should  be  prepared  just  before  use.     Three  fluids 

1 NH2  NH2 

/C6H4  —  V-^6H4y^ 
CH3  CH3 

*  Boas:  Deul.  med.  Woch.,  37,  62,  1911. 

'  Boas  suggests  using  an  ether  extract  of  the  fecal  suspension  thus  eliminating  the 
necessity  of  boiling.  However,  oxidizing  enzymes  are  the  main  sources  of  error  here  and  the 
action  is  easily  and  effectively  eliminated  by  boiling.  (See  White:  Boston  Medical  and 
Surgical  Journal,  164,  876,  191 1.) 

*  Prepared  by  dissolving  1-2  grams  of  phenolphthalein  and  25  grams  of  KOH  in  100  c.c. 
of  distilled  water.  Add  10  grams  of  powdered  zinc  and  heat  gently  until  the  solution  is 
decolorized.     Prepared  in  this  way  the  solution  will  not  deteriorate  on  standing. 

*  Deiitsch.  med.  Woch.,  Aug.  6,  1914. 
'  Wien.  med.  Woch.,  Sept.  5,  1914. 
''Lyon  Med.,  46,  313,  1914. 

*  Hematein  is  a  brownish-red  crystalline  substance  derived  from  hematoxylin  by  the 
successive  action  of  ammonia  and  acetic  acid.  It  should  not  be  confused  with  hematin, 
the  hemoglobin  derivative. 


FECES  235 

are  required:  (i)  a  0.05  per  cent  aqueous  solution  of  hematein;  (2)  a 
40  per  cent  solution  of  sodium  hydroxide  and  (3)  3  per  cent  hydrogen 
peroxide.     These  will  keep  almost  indefinitely. 

The  test  may  be  performed  as  follows :  Take  4-5  c.c.  of  the  liquid  specimen  in  a 
tube  and  in  another  tube  take  the  same  amount  of  material  known  not  to  contain 
blood  as  a  control.  To  each  add  4-5  c.c.  of  the  sodium  hydroxide  solution  and 
shake.  Then  to  each  of  the  tubes  add  2  drops  of  the  hematein  solution.  A  blue 
color  of  about  equal  intensity  will  develop  in  both  tubes.  Then  add  10  drops  of 
hydrogen  peroxide  to  each  tube  and  compare.  If  blood  is  present,  the  tube  con- 
taining it  will  turn  very  rapidly  (in  three  or  four  seconds;  to  violet  red,  then  in 
twenty  seconds  to  clear  brown,  in  forty  seconds  to  pale  yellow  while  the  second 
tube  will  not  show  these  changes  for  several  minutes.  The  reaction  is  said  to 
detect  blood  when  present  in  a  concentration  of  i  part  in  400,000. 

(e)  Aloin-turpenlive  Test. — Mix  the  stool  very  thoroughly  and  take  about 
5  grams  of  the  mixture  for  the  test.  Reduce  this  sample  to  a  semi-fluid  mass  by 
means  of  distUled  water  and  extract  very  thoroughly  with  an  equal  volimie  of  ether 
to  remove  any  fat  which  may  be  present.  Now  treat  the  extracted  feces  with  one- 
third  its  volume  of  glacial  acetic  acid  and  10  c.c.  of  ether  and  extract  ver>'  thoroughly 
as  before.     The  acid-ether  extract  wUl  rise  to  the  top  and  may  be  removed. 

Introduce  2-3  c.c.  of  this  acid-ether  solution  into  a  test-tube,  add  an  equal 
volume  of  a  dilute  solution  of  aloin  in  70  per  cent  alcohol  and  2-3  c.c.  of  ozonized 
turpentine  and  shake  the  tube  gently.  If  blood  is  present  the  entire  volume  of  fluid 
ordinarily  becomes  pink  and  finally  cherry  red.  In  some  instances  the  color  wiU 
be  limited  to  the  aloin  solution  which  sinks  to  the  bottom.  This  color  reaction 
should  occur  within  15  minutes  in  order  to  indicate  a  positive  test  for  blood,  since 
the  aloin  will  turn  red  of  itself  if  allowed  to  stand  for  a  longer  period.  The  color  is 
ordinarily  light  yellow  in  a  negative  test.  Hydrogen  peroxide  is  not  a  satisfactor>- 
substitute  for  turpentine  in  the  test. 

(f  j  Cowie's  Guaiac  Test. — To  i  gram  of  moist  feces  add  4-5  c.c.  of  glacial 
acetic  acid  and  extract  the  mixture  with  30  c.c.  of  ether.  To  1-2  c.c.  of  the  extract 
add  an  equal  volume  of  water,  agitate  the  mixture,  introduce  a  few  granules  of 
powdered  guaiac  resin,  and  after  bringing  the  resin  into  solution,  gradually  add 
30  drops  of  old  turpentine  or  hydrogen  peroxide.  A  blue  color  indicates  the 
presence  of  blood.  Cowie  claims  that  by  means  of  this  test  an  intestinal  hemor- 
rhage of  I  gram  can  easily  be  detected  by  an  examination  of  the  feces. 

(g)  Weber's  Guaiac  Test. — Mix  a  little  feces  with  30  per  cent  acetic  acid  to  form 
a  fluid  mass.  Transfer  to  a  test-tube  and  extract  with  ether.  If  blood  is  present 
the  ether  will  assume  a  brownish-red  color.  Filter  off  the  ether  extract  and  to  a 
portion  of  the  filtrate  add  an  alcoholic  solution  of  guaiac  (strength  about  1:60),' 
drop  by  drop,  until  the  fluid  becomes  turbid.  Now  add  hydrogen  peroxide  or  old 
turpentine.     In  the  presence  of  blood  a  blue  color  is  produced  (see  page  2  58). 

(A)  Acid-hematin. — Examine  some  of  the  ethereal  extract  from  Experiment  (g) 
spectroscopically.  Note  the  typical  spectrum  of  acid-hematin  (see  Absorption 
Spectra.  Plate  II). 

7.  HydrobiUrubin.  Schmidt's  Test.  Rub  up  a  small  amount  of  feces  in  a 
mortar  with  a  concentrated  aqueous  solution  of  mercuric  chloride.     Transfer  to  a 

^  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin. 


236  PHYSIOLOGICAL    CHEMISTRY 

shallow,  flat-bottomed  dish  and  allow  to  stand  6-24  hours.  The  presence  of 
hydrobilirubin  will  be  indicated  by  a  deep  red  color  being  imparted  to  the  par- 
ticles of  feces  containing  this  pigment.  This  red  color  is  due  to  the  formation 
of  hydrobihrubin-mercury.  If  unaltered  bihrubin  is  present  in  any  portion  of 
the  feces  that  portion  will  be  green  in  color  due  to  the  oxidation  of  biUrubin  to 
biUverdin. 

Another  method  for  the  detection  of  hydrobilirubin  is  the  following:  Treat 
the  dry  feces  with  absolute  alcohol  acidified  with  sulphuric  acid  and  shake 
thoroughly.  The  acidified  alcohol  extracts  the  pigment  and  assumes  a  reddish 
color.  Examine  a  little  of  this  fluid  spectroscopically  and  note  the  typical 
spectrum  of  hydrobilirubin  (Absorption  Spectra,  Plate  II). 

8.  BiUrubin.  1  (a)  Gmelin's  Test. — Place  a  few  drops  of  concentrated 
nitric  acid  in  an  evaporating  dish  or  on  a  porcelain  test-tablet  and  allow  a  few 
drops  of  the  feces  and  water  to  mix  with  it.  The  usual  play  of  colors  of  Gmelin's 
test  is  produced,  i.e.,  green,  blue,  violet,  red,  and  yellow.  If  so  desired,  this 
test  may  be  executed  on  a  slide  and  observed  under  a  microscope. 

(b)  Htippert's  Test. — Treat  the  feces  with  water  to  form  a  semi-fluid  mass,  add 
an  equal  amount  of  milk  of  lime,  shake  thoroughly,  and  filter.  Wash  the  precipi- 
tate with  water,  then  transfer  both  the  paper  and  the  precipitate  to  a  small  beaker 
or  flask,  add  a  small  amount  of  95  per  cent  alcohol  acidified  slightly  with  sulphuric 
acid,  and  heat  to  boiling  on  a  water-bath.  The  presence  of  bilirubin  is  indicated 
by  the  alcohol  assuming  a  green  color. 

Steensma  advises  the  addition  of  a  drop  of  a  0.5  per  cent  solution  of  sodium 
nitrite  to  the  acid-alcohol  mixture  before  warming  on  the  water-bath.  Try  this 
modification  also. 

9.  Bile  Acids. — Extract  a  small  amount  of  feces  with  alcohol  and  filter. 
Evaporate  the  filtrate  on  a  water-bath  to  drive  off  the  alcohol  and  dissolve  the 
residue  in  water  made  sUghtly  alkaline  with  potassixim  hydroxide.  Upon  this 
aqueous  solution  try  any  of  the  tests  for  bile  acids  given  on  page  208. 

10.  Casein. — Extract  the  fresh  feces  first  with  a  dilute  solution  of  sodium 
chloride,  and  later  with  water  acidified  with  dilute  acetic  acid,  to  remove  soluble 
proteins.  Now  extract  the  feces  with  0.5  per  cent  sodium  carbonate  and  filter. 
Add  dilute  acetic  acid  to  the  filtrate  to  precipitate  the  casein,  being  careful 
not  to  add  an  excess  of  the  reagent  as  the  casein  would  dissolve.  Filter  off  the 
casein  and  test  it  according  to  directions  given  on  page  321.  Casein  is  found 
principally  in  the  feces  of  children  who  have  been  fed  a  milk  diet.  Mucin  would 
also  be  extracted  by  the  dilute  alkali,  if  present  in  the  feces.  What  test  could 
you  make  on  the  newly  precipitated  body  to  differentiate  between  mucin  and 
casein? 

11.  Nucleoprotein. — Mix  the  stool  thoroughly  with  water,  transfer  to  a  flask, 
and  add  an  equal  amount  of  saturated  lime  water.  Shake  frequently  for  a  few 
hours,  filter,  and  precipitate  the  nucleoprotein  with  acetic  acid.  Filter  off  this 
precipitate  and  test  it  as  follows: 

(o)  Phosphorus. — Test  for  phosphorus  by  fusion  (see  page  129). 
(ft)  Solubility. — Try  the  solubility  in  the  ordinary  solvents. 

(c)  Protein  Color  Test. — Try  any  of  the  protein  color  tests. 

^  The  detection  of  bilirubin  in  the  feces  is  comparatively  simple  provided  it  is  not  ac- 
companied by  other  pigments.  When  other  pigments  are  present,  however,  it  is  difficult  to 
detect  the  bilirubin  and,  at  times,  may  be  found  impossible. 


FECES  237 

What  proof  have  you  that  the  above  body  was  not  mucin?  What  other  test 
can  you  use  to  differentiate  between  nucleoprotein  and  mucin? 

12.  Albumin  and  Globulin. — Extract  the  fresh  feces  with  a  dilute  solution  of 
sodium  chloride.  (The  preliminary  extract  from  the  preparation  of  casein  (10), 
above,  may  be  utilized  here.)  Filter,  and  saturate  a  portion  of  the  filtrate  with 
sodium  chloride  in  substance.  A  precipitate  signifies  globulin.  Filter  off  the  pre- 
cipitate and  acidify  the  filtrate  slightly  with  dilute  acetic  acid.  A  precipitate  at 
this  point  signifies  albumin.     Make  a  protein  color  test  on  each  of  these  bodies. 

13.  Proteose  and  Peptone, — Heat  to  boiling  the  portion  of  the  sodium  chloride 
extract  not  used  in  the  last  experiment.  Filter  oflf  the  coagulum,  if  any  forms. 
Acidify  the  filtrate  slightly  with  acetic  acid  and  saturate  with  sodium  chloride  in 
substance.  A  precipitate  here  indicates  proteose.  Filter  it  ofif  and  test  it  according 
to  directions  given  on  page  120.     Test  the  filtrate  for  peptone  by  the  biuret  test. 

14.  Inorganic  Constituents. — Incinerate  a  smaU  amount  of  feces  in  a  crucible 
and  dissolve  the  ash  in  a  small  volume  of  dilute  nitric  acid.  Dilute  with  water  and 
filter.     Make  the  following  tests  upon  the  clear  filtrate. 

(c)  Chlorides. — Acidify  with  nitric  acid  and  add  silver  nitrate. 

{h)  Phosphates. — Acidify  with  nitric  acid,  add  molybdic  solution,  and  warm 
gently. 

(c)  Sulphates. — Acidify  with  hydrochloric  acid,  add  barium  chloride,  and  warm. 

{d)  Calcium. — Neutralize  with  ammonium  hydroxide,  make  slightly  acid  with 
acetic  acid  and  add  ammonium  oxalate.    Let  stand. 

(e)  Magnesium. — Neutralize  with  ammonium  hydroxide,  and  add  Na2HP04 
and  excess  of  NH4OH.    Let  stand. 

15.  Indole  Reactions. — Rub  up  the  stool  with  water  to  form  a  thin  paste 
and  distill  first  in  alkaline  and  then  in  acid  solution.  Test  the  distillate  by 
any  of  the  tests  for  the  detection  of  indole  in  putrefaction  mixtures  (see 
page  218). 

16.  Schmidt's  Nuclei  Test. — This  test  serves  as  an  aid  to  the  diag- 
nosis of  pancreatic  insufficiency.  The  test  is  founded  upon  the  theory 
that  cell  nuclei  are  digestible  only  in  pancreatic  juice,  and  therefore 
that  the  appearance  in  the  feces  of  such  nuclei  indicates  insufficiency  of 
pancreatic  secretion. 

The  procedure  is  as  follows:  Cubes  of  fresh  beef  about  12  cm.  square  are 
enclosed  in  small  gauze  bags  and  ingested  with  a  test  meal.  Subsequently 
the  fecal  mass  resulting  from  this  test  meal  is  examined,  the  bag  opened,  and 
the  condition  of  the  enclosed  residue  determined.  Under  normal  conditions  the 
nuclei  would  be  digested.  Therefore  if  the  nuclei  are  found  to  be  for  the  most 
part  undigested,  and  the  intervening  period  has  been  sufficient  to  permit  of  the 
full  activity  of  the  pancreatic  function  (at  least  six  hours),  it  may  be  considered  a 
sign  of  pancreatic  insufficiency  (see  Fig.  69,  p.  231). 

It  has  been  claimed  by  Steele  that  under  certain  conditions  the  non- 
digestion  of  the  nuclei  may  indicate  a  general  lowering  of  the  digestive 
power  rather  than  a  true  pancreatic  insufficiency. 

Kashiwado^  has  suggested  the  use  of  stained  cell  nuclei  in  this  test. 
A  preparation  put  out  under  the  name  "Gefarbte  gewebskerne  zur 

^  Kashiwado:  Deut.  Arch.  Klin.  Med.,  104,  584,  191 1. 


238 


PHYSIOLOGICAL   CHEMISTRY 


Pankreasfuntionspriifung  nach  Prof.  Dr.  Schmidt  und  Dr.  Kashiwado" 
consists  of  a  mixed  preparation  of  thymus  cells,  the  nuclei  of  which 
are  stained  by  iron  hematoxylin,  and  lycopodium  powder.  After 
administration,  the  lycopodium,  which  is  readily  recognized,  is  sought 
for  in  the  stool  and  when  found  that  portion  is  examined  for  the  stained 
thymus  cells.  Their  statement  is  as  follows:  If  stained  nuclei  are 
not  found  in  the  feces  after  an  intestinal  transit  of  sufficient  duration 
(at  least  six  hours)  normal  pancreatic  function  (external)  is' indicated. 
If,  however,  all  or  part  of  the  cells  are  found,  a  definite  disturbance  in 
pancreatic  function  is  present. 

17.  Influence  of  Drugs  upon  the  Color  of  the  Stool. — Ingest  an  ordinary 
mixed  diet,  take  the  indicated  dose  of  one  of  the  following  drugs,  "separate" 
the  feces  (see  page  587)  and  after  the  "marker"  appears  note  the  color  of  the 
stools  evacuated : 


Drug 

Dose 

Color  of  stool 

1  Bismuth  subnit'rate,  grams 

5 

Black. 

Calomel,  mg 

130-140 

Green. 

Reduced  iron,  mg 

65-70      Grayish  black  turning  darker  on  exposure  to  air. 

1 
Methylene  blue,  mg 130-140,  Blue,  especially  after  exposure  to  air. 

Manganese  dioxide,  mg. . . . 

130-1401  Dark  brown  or  black. 

Hematoxylin,  grams 

I 

Reddish  brown. 

Rhubarb,  c.c.  fluid  extract.. 

1 

2       '  Yellow. 

Senna,  c.c.  fluid  extract. . . 

4         Dark  yellow. 

Cambogia,  mg 

130-140    Dark  yellow. 

Santonin,  mg 

6';-70       Dark  vellow. 

1 

18.  Einhom's  Bead  Test.* — ^This  is  a  method  for  testing  the  digestive  func- 
tion. In  some  respects  it  is  similar  to  SahU's  desmoid  reaction  (see  Gastric 
Analysis).  The  procedure  consists  in  wrapping  the  material  under  examination 
(catgut,  fish-bone,  raw  beef,  cooked  potatoes,  thjrmus  gland  or  mutton  fat,  etc.) 
in  gauze  to  which  glass  beads  of  various  colors  are  attached  and  enclosing  gauze 
and  beads  in  a  gelatine  capsule.^  The  gelatine  capsule  is  swallowed  and  the 
beads  serve  to  faciUtate  the  separation  of  the  gauze  from  the  feces.    The  residue 

^  Einhorn:  The  Post-Graduale,  May,  191 2:  Boas^  Arch.,  12,  26,  1906;  13,  35,  1907;  Ibid., 
47S;  IS,  part  2,  1909. 

^  Ordinarily  two  substances  are  attached  to  each  bead,  three  beads  tied  together  and 
enclosed  in  one  capsule.    Test  capsules  may  be  obtained  from  Eimer  and  Amend,  New  York. 


FECES  239 

within  the  gauze  is  then  examined.  If  beads  appear  in  much  less  than  24  hours 
an  accelerated  motility  is  indicated,  whereas  an  interval  of  48  hours  or  over 
elapsing  indicates  retarded  motihty.  If  gastric  function  alone  is  to  be  studied 
silk  threads  are  attached  to  the  beads  and  the  latter  are  withdrawn  and  examined 
before  they  have  passed  into  the  intestine. 

19.  "Separation"  of  Feces. — In  order  to  become  famiUar  with  the  method 
ordinarily  utihzed  in  metaboUsm  experiments  to  differentiate  the  feces  which 
correspond  to  the  food  ingested  during  any  given  interval,  and  at  the  same  time 
to  secure  data  as  to  the  length  of  time  necessary  for  ingested  substances  to  pass 
through  the  alimentary  tract  proceed  as  follows:  Just  before  one  of  the  three 
meals  of  the  day  ingest  a  gelatine  capsule  (No.  00)  containing  0.2  0.3  of  a  gram 
of  carmine  or  charcoal.  Make  an  Inspection  of  all  stools  subsequently  dropped 
and  note  the  time  interval  elapsing  between  the  ingestion  of  the  capsule  and  the 
appearance  of  its  contents  in  the  feces.  Under  normal  conditions  this  period  is 
ordinarily  24  hours.     This  test  is  thus  an  index  of  intestinal  motiUty. 

20.  Influence  of  Foods  upon  the  Color  of  the  Stool. — Ingest  a  diet  which 
contains  a  Uberal  quantity  of  one  of  the  following  articles  of  diet,  "separate"  the 
feces  (page  587)  and  after  the  "marker"  appears  note  the  color  of  the  stools 
evacuated : 


Article  of  diet 

Color  of  stool 

Milk. 

Light  yellow  or  grayish  white. 

Meat 

Brownish  black. 

Chlorophyllic  vegetables,  e.g.,  spinach — 

Greenish.                                                             | 

Non-rhlorophyllic  vegetables 

Light  brown. 

Cherries  or  blackberries 

Reddish  brown. 

Cocoa 

Dark  red  or  chocolate  brown. 

Coffee 

Dark  brown. 

Corn  meal 

Light  colored. 

21.  Quantitative  Determination  of  Fecal  Amylase  (The  Author's^  Modification 
of  Wohlgemuth's-  Method). — Weigh  accurately  about  2  grams  of  fresh  feces  into 
a  mortar,'  add  8  c.c.  of  a  phosphate-chloride  solution  (o.i  mol  dihydrogen  sodium 
phosphate  and  0.2  mol  disodium  hydrogen  phosphate  per  liter  of  i  per  cent  sodium 
chloride),  2  c.c.  at  a  time,  rubbing  the  feces  mixture  to  a  homogeneous  consistency 
after  each  addition  of  the  extraction  medium.  Permit  the  mixture  to  stand  at 
room  temperature  for  a  half-hour  with  frequent  stirring.  We  now  have  a  neutral 
fecal  suspension.  Transfer  this  suspension  to  a  15  c.c.  graduated  centrifuge  lube, 
being  sure  to  wash  the  mortar  and  pestle  carefully  with  the  phosphate-chloride 
solution  and  add  all  washings  to  the  suspension  in  the  centrifuge  tube.  The  sus- 
pension is  now  made  up  to  the  15  c.c.  mark  with  the  phosphate-chloride  solution 

1  Hawk:  Arch.  Int.  Med.,  8,  552,  191 1. 

^  Wohlgemuth:  Bcrl.  klin.  Wocli.,  47,  3,  92,  1910;  also  see  page  192,  this  book. 

'  Duplicate  determinations  should  be  made. 


240  PHYSIOLOGICAL    CHEMISTRY 

and  centrifugated  for  a  15-niinute  period,  or  longer  if  necessary,  to  secure  satis- 
factory' sedimentation.  At  this  point,  read  and  record  the  height  of  the  sediment 
column.  Remove  the  supernatant  liquid  by  means  of  a  bent  pipette,  transfer  it  to 
a  50  CO.  volumetric  flask  and  dilute  it  to  the  50  c.c.  mark  with  the  phosphate- 
chloride  solution.  Mix  the  fecal  extract  thoroughly  by  shaking  and  determine  its 
amylolytic  activity.  For  this  purpose  a  series  of  six  graduated  tubes  is  prepared, 
containing  volumes  of  the  extract  ranging  from  2.5  c.c.  to  0.078  c.c.  Each  of  the 
intermediate  tubes  in  this  series  will  thus  contain  one-half  as  much  fluid  as  the 
preceding  tube.  Now  make  the  contents  of  each  tube  2.5  c.c.  by  means  of  the 
phosphate-chloride  solution  in  order  to  secure  a  uniform  electrolyte  concentration. 
Introduce  5  c.c.  of  a  i  per  cent  soluble  starch  solution^  and  three  drops  of  toluol 
into  each  tube,  thoroughly  mix  the  contents  by  shaking,  close  the  tubes  by  means 
of  stoppers  and  place  them  in  an  incubator  at  38°C.  for  24  hours.  At  the  end  of 
this  time  remove  the  tubes,  fill  each  to  within  half  an  inch  of  the  top  with  ice-water, 
add  I  drop  of  tenth-normal  iodin  solution,  thoroughly  mix  the  contents  and  examine 
the  tubes  carefully  with  the  aid  of  a  strong  light.  Select  the  last  tube  in  the  series 
which  shows  entire  absence  of  blue  color,  thus  indicating  that  the  starch  has  been 
completely  transformed  into  dextrin  and  sugar,  and  calculate  the  amylolytic  activity 
on  the  basis  of  this  dilution.  In  case  of  indecision  between  two  tubes,  add  an  extra 
drop  of  the  iodin  solution  and  observe  them  again.^ 

The  amylolytic  value,  Df,  of  a  given  stool,  may  be  expressed  in  terms  of  i  c.c. 
of  the  sediment  obtained  by  centrifugation  as  above  described.  For  example,  if  it 
is  found  that  0.31  c.c.  of  the  phosphate-chloride  extract  of  the  stool  acting  at  38°C. 
for  24  hours  completely  transformed  the  starch  in  5  c.c.  of  a  i  per  cent  starch  solu- 
tion, then  we  would  have  the  following  proportion: 

0.31  :5  (c.c.  starch)  ::  i(c.c.  extract)  :X 

The  value  of  X  in  this  case  is  16. i,  which  means  that  i  c.c.  of  the  fecal  extract 
possesses  the  power  of  completely  digesting  16. i  c.c.  of  a  i  per  cent  starch  solution 
in  24  hours  at  38°C. 

Inasmuch  as  stools  vary  so  greatly  as  to  water  content,  it  is  essential  to  an 
accurate  comparison  of  stools  that  such  comparison  be  made  on  the  basis  of  the 
solid  matter.  Supposing,  for  example,  that  in  the  above  determination  we  had 
6.2  c.c.  of  sediment.     Since  the  supernatant  fluid  was  removed  and  made  up  to 

1  In  preparing  the  i  per  cent  solution,  the  weighed  starch  powder  should. be  dissolved 
in  cold  distUled  water  in  a  casserole  and  stirred  untU  a  homogeneous  suspension  is  obtained. 
The  mixture  should  then  be  heated  with  constant  stirring,  unril  it  is  clear.  This  ordinarily 
takes  from  eight  to  ten  minutes.  A  slightly  opaque  solution  is  thus  obtained,  which 
should  be  cooled  and  made  up  to  the  proper  volume  before  using. 

2  Theoretically  we  would  expect  the  colors  to  range  from  a  light  yellow  to  a  dark  blue, 
with  red  tubes  holding  an  intermediate  position  in  the  series.  This  color  sequence  does 
often  occur,  but  its  occurrence  is  far  from  universal.  Many  times  the  first  tubes  in  the  series, 
i.e.,  those  containing  the  largest  quantities  of  the  fecal  extract,  will  exhibit  a  bluish  cast  of 
color  which  should  not  be  confused  with  the  starch  color  reaction.  When  these  blue  tubes 
are  present,  they  are  generally  followed  by  yellow,  red  and  blue  tubes  in  order,  the  final  blue 
tube,  of  course,  being  the  regulation  starch  reaction.  Occasionally  greenish  colors  will  be 
obtained  to  the  left  of  the  red  color.  It  also  sometimes  happens  that  it  is  somewhat  dif- 
ficult to  determine  in  which  tube  to  the  right  of  the  red  color  the  starch  blue  color  is  first  de- 
tected, unless  the  tube  be  examined  carefully  before  a  strong  light.  In  every  instance,  how- 
ever, \yhen  these  blue  and  green  colors  are  observed,  it  is  noted  that  tubes  possessing  the 
true  dextrin  red  color  are  always  present  between  these  tubes  and  the  tubes  possessing  the 
true  starch  blue  color.  It  is  evident,  therefore,  that  these  bluish  tints  in  the  tubes  to  the 
left  of  the  dextrin  color  cannot  be  due  to  the  presence  of  starch.  The  cause  of  the  blue  color 
reaction  in  the  first  tubes  of  the  series  has  not  been  ascertained  as  yet. 


PECES  241 

50  c.c.  before  testing  its  amylolytic  value,  it  is  evident  that  i  c.c.  of  this  sediment  is 
equivalent  to  8.1  c.c.  of  extract.  Therefore,  in  order  to  derive  the  amylolytic 
value  of  I  c.c.  of  sediment,  we  must  multiply  the  value  (16. i)  as  obtained  above 
for  the  extract,  by  8.1.  This  yields  130.4  and  enables  us  to  express  the  activity 
as  follows: 

Dl24h  =   130-4 

The  above  method  of  calculation  is  that  suggested  by  Wohlgemuth.  In  case  time 
and  facilities  permit  cf  the  determination  of  the  moisture  content  of  the  feces,  it  is 
much  more  accurate  and  satisfactory  to  place  the  amylolytic  values  of  the  stools 
on  a  "gram  of  dry  matter"  basis.  The  amylolytic  values  of  the  stools  are  expressed 
as  the  number  of  cubic  centimeters  of  i  per  cent  starch  solution  which  the  amylase 
content  of  i  gram  of  dry  feces  is  capable  of  digesting. 

22.  Quantitative  Determination  of  Fecal  Bacteria.^ — The  method  is  a  simpli- 
fication of  MacXeal's  adaptation  of  the  Strasburger  procedure. ^  About  2  grams  of 
feces  are  accurately  weighed  and  placed  in  a  50  c.c.  centrifuge  tube.  To  the  feces 
in  the  tube  a  few  drops  of  0.2  per  cent  hydrochloric  acid  are  added,  and  the  material 
is  mixed  to  a  smooth  paste  by  means  of  a  glass  rod.  Further  amounts  of  the  acid 
are  added  with  continued  crushing  and  stirring  until  the  material  is  thoroughly 
suspended.  The  tube  is  then  whirled  in  the  centrifuge  at  high  speed  for  one-half 
to  one  minute.  The  suspension  is  found  sedimented  into  more  or  less  definite 
layers,  the  uppermost  of  which  is  fairly  free  from  the  larger  particles.  The  upper 
and  more  liquid  portion  of  the  suspension  is  now  drawn  off  by  means  of  a  pipette 
and  transferred  to  a  beaker.^  The  sediment  remaining  in  the  tube  is  again  rubbed 
up  with  the  glass  rod  with  the  addition  of  further  amounts  of  dilute  acid,  and  again 
centrifugalized  for  one-half  to  one  minute.  The  supernatant  liquid  is  pipetted  off 
and  added  to  the  first,  the  same  pipette  being  used  for  the  one  determination  through- 
out.'* A  third  portion  of  the  dilute  acid  is  then  added  to  the  sediment,  which  is 
again  mixed  by  stirring  and  again  centrifugalized.  All  the  washings  are  added  to 
the  first  one,  and  during  the  process  care  is  taken  to  wash  the  material  from  the 
walls  and  mouth  of  the  centrifuge  tube  down  into  it.  Finally,  when  the  sediment 
is  sufficiently  free  from  bacteria,  the  various  remaining  particles  are  visibly  clean, 
and  the  supernatant  liquid  after  centrifugalization  remains  almost  clear.  This  is 
removed  to  the  beaker  in  which  are  now  practically  all  the  bacteria  present  in  the 
original  portion  of  feces,  together  with  some  solid  matter  not  yet  separated.  In  the 
centrifuge  tubes  there  is  a  considerable  amount  of  bacferia-free  solid  matter. 

The  suspension  is  now  transferred  to  the  same  centrifuge  tube,  centrifugalized 
for  a  minute,  and  the  supernatant  liquid  transferred  to  a  clean  beaker  by  means  of 
the  same  pipette.  The  tube  is  then  refilled  from  the  first  beaker  and  thus  all  the 
suspension  centrifugalized  a  second  time.  The  beaker  is  finally  carefully  washed 
with  the  aid  of  a  rubber-tipped  glass  rod,  the  second  sediment  in  the  centrifuge 
tube  is  washed  free  of  bacteria  by  means  of  this  wash  water  and  by  successive  por- 
tions of  the  dilute  acid,  and  the  supernatant  liquid  after  centrifugalization  is  added 
to  the  contents  of  the  second  beaker.     The  second  clean  sediment  is  added  to  the 

^  Mattill  and  Hawk:  Jour.  Exp.  Med.,  14,  433,  191 1. 

^  MacNeal,  Latzer  and  Kerr,  Joiir.  Inf.  Dis.,  6,  123,  1909. 

'  A  25  c.c.  pipette  is  the  most  satisfactory  size;  to  facilitate  observation,  the  delivery  tube 
is  bent  near  the  bulb  to  an  angle  of  about  120  degrees. 

^  A  convenient  support  for  the  pipettes  is  a  wire  spring  on  a  glass  base,  such  as  is  used  on 
a  desk  for  pen-holders.     The  delivery  tube,  just  where  it  is  bent,  is  inserted  between  the 
wires,  and  any  hquid  not  delivered  collects  in  the  bend  of  the  tube. 
16 


242  PHYSIOLOGICAL   CHEMISTRY 

first.  The  bacterial  suspension  now  in  the  second  beaker  is  again  centrifugalized 
in  the  same  way  and  a  third  portion  of  bacteria-free  sediment  is  separated.  Fre- 
quently a  fourth  serial  centrifugalization  is  performed — always  if  the  third  sediment 
is  of  appreciable  quantity.  At  all  stages  of  the  separation,  small  portions  of  the 
dilute  hydrochloric  acid  are  used,  so  that  the  final  suspension  shall  not  be  too  vo- 
luminous. Ordinarily  it  amounts  to  125  to  200  c.c.  At  the  same  time,  the  final 
amount  of  fluid  should  not  be  too  small,  as  shown  by  Ehrenpfordt,^  because  the 
viscosity  accompanying  increased  concentration  prevents  proper  and  complete 
sedimentation. 

To  the  final  bacterial  suspension  an  equal  volume  of  alcohol  is  added  and  the 
beaker  set  aside  to  concentrate.  A  water-bath  at  50°  to  6o°C.  is  very  satisfactory. 
After  two  or  three  days,  when  the  liquid  is  concentrated  to  about  50  c.c,  the  beaker 
is  removed  and  about  200  c.c.  of  alcohol  are  added.  The  beaker  is  covered  and 
allowed  to  stand  at  room  temperature  for  24  hours.  At  the  end  of  this  time  the 
bacterial  substance  is  generally  settled,  so  that  most  of  the  clear  supernatant  liquid, 
of  dark  brown  color,  can  be  directly  siphoned  off  without  loss  of  solid  matter.  The 
remainder  is  then  transferred  to  centrifuge  tubes,  centrifugalized,  and  the  remaining 
clear  liquid  pipetted  off."  The  sediment  consists  of  the  bodies  of  the  bacteria,  and 
is  transferred  to  a  Kjeldahl  flask  for  nitrogen  determination.  This  is  the  bacterial 
nitrogen.  Where  a  determination  of  bacterial  dry  substance  is  desired,  the  sedi- 
ment of  bacteria  is  extracted  by  absolute  alcohol  and  ether  in  succession,  trans- 
ferred to  a  weighed  porcelain  crucible,  and  dried  at  io2°C.  to  constant  weight. 
This  dried  sample  is  then  used  in  the  nitrogen  determination.  Our  procedure 
differs  from  that  of  MacNeal  in  that  the  bacterial  dry  matter  is  not  determined. 
A  saving  of  about  seven  days'  time  and  of  considerable  labor  is  accomplished  by 
this  omission. 

Inasmuch  as  it  has  been  shown  by  various  investigators  that  such  bacteria  as 
are  present  in  the  feces  contain  on  the  average  about  11  per  cent  of  nitrogen,  the 
values  for  bacterial  nitrogen  as  determined  by  our  method  may  conveniently  serve 
as  a  basis  for  the  calculation  of  the  actual  output  of  bacterial  substance. 

23.  Quantitative  Deterinination  of  Indol  in  Feces.  Bergeim's  Modification  of  the 
Herter -Foster  Method.' — Principle. — The  feces  is  distilled  from  alkaline  solution 
to  remove  phenols.  This  distillate  are  again  distilled  from  acid  solution  to  remove 
ammonia.  The  indol  in  the  final  distillate  is  treated  with  /3-naphthaquinone  sodium 
monosulphonate  and  alkali  and  the  blue  compound  formed  extracted  with  chloro- 
form and  determined  colorimetrically. 

Procedure. — Rub  30-50  grams  of  the  fresh,  well-mixed  feces  in  a  mortar  with 
water  to  a  uniform  consistency.  Transfer  to  a  wide  mouth  Kjeldahl  flask  of  about 
1000  c.c.  capacity,  rinsing  mortar  and  neck  of  flask  with  distilled  water  to  make 
about  400  c.c.  Add  5  c.c.  of  10  per  cent  KOH  solution  and  about  2  c.c.  of  paraffin 
to  decrease  foaming.  Distill  with  steam  using  ordinary  Kjeldahl  distillation  ap- 
paratus with  good  stream  of  water  in  the  condenser.  Heat  carefully  for  a  few 
minutes  until  danger  of  foaming  is  past  and  then  allow  to  boil  vigorously.  Distill 
over  500  c.c.  of  liquid,  bringing  the  volume  of  the  fecal  suspension  down  to  about 
100  c.c.  toward  the  end  of  the  distillation. 

^  Ehrenpfordt:  Zeii.  cxp.  Path.  Thcr.,  7,  455,  1909. 

2  In  later  work  (see  Blatherwick  and  Hawk:  Biochem.  Bull.,  3,  28,  1913)  it  was 
found  advantageous  to  centrifugalize  with  alcohol  and  ether  in  succession  before  trans- 
ferring the  bacterial  cells  to  Kjeldahl  flasks. 

3  Herter  and  Foster:  Jour.  Biol.  Chcm.,  i,  257,  1906.  Bergeim,  Fishback,  and  Hawk: 
Unpublished  data. 


FECES 


243 


Transfer  the  distillate  to  a  clean  Kjeldahl  flask,  add  2  drops  of  phenolphthalein 
as  an  indicator.  Make  neutral  with  N  sulphuric  acid  and  add  i  c.c.  excess.  Dis- 
till with  steam  as  before,  collecting  the  first  500  c.c.  of  distillate  and  bringing  the 
residue  finally  to  about  100  c.c.     Mix  distillate  well  by  shaking. 

Take  an  aliquot  portion  of  the  distillate  (100  c.c);  add  i  c.c.  of  a  2  per  cent 
solution  of  /3-naphthaquinone  sodium  monosulphonate  solution.  Then  add  2  c.c. 
of  10  per  cent  KOH.  Shake  and  let  stand  for  15  minutes.  This  is  best  carried  out 
in  a  150  c.c.  Squibb  shape  separatory  funnel.  Extract  with  chloroform,  shaking 
vigorously,  using  a  10  c.c.  and  a  7  c.c.  portion  which  will  bring  the  total  volume  of 
the  extract  to  the  mark  of  a  15  c.c.  graduated  tube.  Mix  thoroughly.  Run  at 
the  same  time  and  in  the  same  way  a  standard  using  i  c.c.  of  a  solution  of  indol  o.i 
mg.  of  indol  per  c.c.  Compare  the  extract  with  this  standard  in  a  colorimeter, 
using  the  standard  ordinarily  at  the  .30-mm.  mark.  Calculate  the  indol  to  the 
basis  of  milligrams  of  indol  per  gram  of  moist  feces. 

Indol.  and  naphthaquinone  solutions  should  be  freshly  prepared  or  may  be 
kept  in  the  ice-box  for  some  days.  Indol  distillates  should  be  kept  in  the  ice-box 
if  not  used  at  once,  especially  in  hot  weather.     The  feces  must  be  fresh. 

24.  Quantitative  Determination  of  Fat  in  Feces. — Principle. — The 
determination  of  fat  in  dried  feces  is  a  more  or  less  tedious  process,  and 
one  which  is  somewhat  dangerous  if  applied  to  pathological  feces.  Most 
of  the  methods  for  the  determination  of  fat  in  the  moist  feces  are  accurate, 
but  require  a  long  time.  Saxon^  has  proposed  a  method  for  the  deter- 
mination of  fat  in  moist  feces,  which  is  speedy,  convenient,  and  accur- 
ate. The  soaps  of  the  feces  are  converted  into  free  fatty  acids  by 
means  of  hydrochloric  acid,  and  the  material  is  then  extracted  by  shak- 
ing with  ether.  The  ether  removes  the  neutral  fat.  the  fatty  acids 
which  were  present  as  such,  the  fatty  acids  derived  from  the  soaps,  and 
the  cholesterol.  The  ether  is  removed  by  distillation,  the  crude  fat 
purified  by  means  of  petroleum  ether,  and  the  weight  of  the  total  fat 
obtained.  The  fat  is  then  dissolved  in  benzol  and  titrated  with  tenth- 
normal sodium  alcoholate  solution,  using  phenolphthalein  as  an  indi- 
cator.    The  fatty  acid  is  calculated,  from  the  titration,  to  stearic  acid. 

Procedure. — Place  about  5  grams  (accurately  weighed  >  of  the  thoroughly 
mixed  feces  in  a  100  c.c.  glass-stoppered  graduated  cylinder. - 

Add  20  c.c.  of  distilled  water,  i  to  2.5  c.c.  of  concentrated  hydrochloric  acid 
(depending  upon  the  amount  of  the  sample)  and  again,  suflBcient  distilled  water 
to  make  a  total  bulk  of  30  c.c.  Add  exactly  20  c.c.  of  ether,  stopper,  and  shake 
vigorously  for  five  minutes.  Allow  to  stand  for  a  few  seconds,  remove  the  stopper, 
add  exactly  20  c.c.  of  95  per  cent  alcohol,  and  again  shake  for  five  minutes. 

Stand  the  cylinder  aside.  The  ether,  containing  practically  all  of  the  fat, 
will  come  to  the  top  as  a  colored  transparent  layer.     Blow  the  ether  layer  ofif  into 

^  Saxon:  J.  Biol.  CItem.,  17,  gg,  19 14. 

^  Care  must  be  taken  not  to  smear  the  neck  of  the  cylinder.  This  may  be  avoided  by 
removing  the  feces  from  the  weighing  bottle  by  means  of  a  glass  rod,  the  end  of  which  is 
flattened,  and  bent  in  the  shape  of  a  hoe,  and  transferring  small  bits  of  the  feces  from  the  hoe 
to  the  cylinder,  using  short  pieces  of  glass  rod,  which  are  dropped  into  the  cylinder  together 
with  the  feces. 


244  PHYSIOLOGICAL   CHEMISTRY 

a  tall  150-200  c.c.  beaker.  ^  The  thin  layer  of  ether  which  remains  is  diluted  with 
5  c.c.  of  ether,  the  tube  slightly  agitated,  and  the  ether  blown  ofif.  This  is  done 
in  all  five  times,  care  being  taken  each  time  to  wash  down  the  sides  of  the  cylinder. 
The  stopper  should  also  be  washed. 

Twenty  c.c.  of  ether  are  again  added,  and  the  cylinder  shaken  for  five  minutes 
and  set  aside.  When  the  ether  has  nearly  stratified,  blow  it  off  and  wash  as 
before.     During  the  second  washing  stratification  will  complete  itself. 

Evaporate  the  ether-  until  no  trace  of  the  alcohol,  which  has  been  carried 
over  with  it,  remains.  To  the  residue  add  30  c.c.  of  low-boiling  petrolevmi  ether 
(should  boil  below  6o°C.),  and  allow  to  stand  over  night.  Petroleimi  for  this 
work  should  be  frequently  tested  for  a  residue  on  evaporation.  If  a  residue  is 
left,  the  ether  shoxild  be  redistilled. 

Filter  the  petroleum  ether  solution  of  the  fat,  catch  the  filtrate  and  washings 
in  a  tall,  weighed,  100  c.c.  beaker,  evaporate  off  the  solvent,  dry  the  beaker  at 
90°C.,  desiccate  and  weigh. 

After  weighing,  dissolve  the  contents  of  the  beaker  in  50  c.c.  of  benzol,  heat 
almost  to  the  boiUng -point,  add  2  drops  of  a  0.5  per  cent  solution  of  phenolphthalein, 
and  titrate  with  a  decinormal  solution  of  sodiimi  alcoholate.^ 

Calculations.- — The  weight  of  total  fat  is  obtained  by  subtracting  the 
weight  of  the  empty  beaker  from  the  weight  of  the  beaker  plus  the  dried 
fat.  The  weight  of  fatty  acids  (in  terms  of  milligrams  of  stearic  acid) 
is  obtained  by  multiplying  the  number  of  cubic  centimeters  of  deci- 
normal sodium  alcoholate  solution  by  the  factor  28.4.  The  difference 
between  the  weight  of  total  fat  and  the  weight  of  fatty  acids  is  the 
weight  of  neutral  fat  in  the  sample  extracted. 

A  separate  determination  without  the  addition  of  hydrochloric  acid 
may  be  run  upon  the  sample,  for  the  purpose  of  determining  the  weight 
of  neutral  fat  and  free  fatty  acids.  The  difference  between  this  weight 
and  the  weight  of  total  fat  is  the  weight  of  fatty  acid  present  in  the 
original  sample  in  the  form  of  soaps. 

^  This  is  accomplished  in  the  same  manner  that  water  is  blown  from  a  wash  bottle. 
The  submerged  end  of  the  delivery  tube  is  bent  upward,  as  in  the  apparatus  used  for  the 
determination  of  fat  in  milk  by  Meig's  method  (see  Milk).  This  avoids  upward  currents 
which  would  disturb  the  subjacent  alcohol-ether-feces  layer. 

^  Erlenmeyer  flasks  of  about  200  c.c.  capacity  may  be  used,  instead  of  beakers,  for  the 
collection  of  the  ether  blown  from  the  cylinders.  The  ether  may  then  be  distilled  and  re- 
covered.    The  same  procedure  may  be  followed  in  removing  the  petroleum  ether. 

^  In  the  preparation  of  the  sodium  alcoholate  solution,  absolute  alcohol  and  freshly  cut, 
bright,  metallic  sodium  are  used;  otherwise  the  procedure  is  the  same  as  that  for  the  stand- 
ardization and  preparation  of  any  alkali  solution. 


CHAPTER  XV 
BLOOD  AND  LYMPH 

Blood  is  composed  of  four  types  of  form-elements  (erythrocytes  or 
red  blood  corpuscles,  leucocytes  or  white  blood  corpuscles,  blood  plates 
or  plaques  and  blood  dust  or  hemoconein)  held  in  suspension  in  a  fluid 
called  hlood  plasma.  These  form-elements  compose  about  60  per  cent  of 
the  blood,  by  weight.  Ordinarily  blood  is  a  dark  red  opaque  fluid  due  to 
the  presence  of  the  red  blood  corpuscles,  but  through  the  action  of 
certain  substances,  such  as  water,  ether,  or  chloroform,  it  may  be 
rendered  transparent.  Blood  so  altered  was  formerly  said  to  be  laked. 
The  term  hemolysis  is  now  used  in  this  connection  and  substances  which 
cause  such  action  are  spoken  of  as  hemolytic  agents.  The  hemolj^tic 
process  is  simply  a  liberation  of  the  hemoglobin  from  the  stroma  of 
the  red  blood  corpuscle.  Normal  blood  is  alkaline  in  reaction  to 
litmus,  the  alkalinity  being  due  principally  to  sodium  carbonate. 
When  examined  according  to  physico-chemical  methods  the  blood  is 
found  to  be  neutral,  i.e.,  it  does  not  contain  an  excess  of  hydroxyl 
ions.  Even  in  cases  of  the  most  pronounced  acidosis  the  reaction  of  the 
blood  is  but  slightly  altered.  The  specific  gravity  of  the  blood  of  adults 
ordinarily  varies  between  1.045  ^^^  i-075-  It  varies  somewhat  with 
the  sex,  the  blood  of  males  having  a  rather  higher  specific  gravity  than 
that  of  females  of  the  same  species.  Under  pathological  conditions 
also  the  density  of  the  blood  may  be  very  greatly  altered.  The  freezing- 
point  (a)  of  normal  blood  is  about  —  o.56°C.  Variations  between 
—  0.51°  and  —  o.62°C.  may  be  due  entirely  to  dietary  conditions,  but  if 
any  marked  variation  is  noted  it  can  in  most  cases  be  traced  to  a 
disordered  kidney  function.  The  total  amount  of  blood  in  the  body  has 
been  variously  estimated  at  from  one-twelfth  to  one-fourteenth  of  the 
body  weight.  Perhaps  1/13.5  is  the  most  satisfactory  figure.  Abder- 
halden  and  Schmidt^  have  suggested  a  unique  method  for  the  deter- 
mination of  this  value.  It  is  based  upon  the  change  in  the  optical 
activity  of  the  blood  upon  injection  of  a  body  of  known  optical  activity, 
such,  for  example,  as  dextrin.  Keith,  Rowntree  and  Geraght>-  have 
recently  made  use  of  a  dye  in  the  determination  of  blood  \olumc. 
.  Among  the  most  important  constituents  of  blood  plasma  are  the  four 

1  Abderhalden  and  Schmidt:  Zeit.  physiol.  chetn.,  66,  120,  1910. 
*  Keith,  Rowntree  and  Geraghty:  Arch.  Int.  Med.,  16,  509,  1915. 

245 


246  PHYSIOLOGICAL    CHEMISTRY 

protein  bodies,  fibrinogen,  nucleoprotein,  serum  globulin  (euglobulin  and 
pseudo-globulin)  and  serum  albumin.  Plasma  contains  about  8.2  per 
cent  of  solids  of  which  the  protein  constituents  named  above  constitute 
approximately  84  per  cent  and  the  inorganic  constituents  (mainly 
chlorides,  phosphates  and  carbonates)  approximately  10  per  cent. 
Among  the  inorganic  constituents  sodium  chloride  predominates.  To 
prevent  coagulation,  blood  plasma  is  ordinarily  studied  in  the  form  of 
an  oxalated  or  salted  plasma.  The  former  may  be  obtained  by  allowing 
the  blood  to  flow  from  an  opened  artery  into  an  equal  volume  of  0.2  per 
cent  ammonium  oxalate  solution,  whereas  in  the  preparation  of  a  salted 
plasma  10  per  cent  sodium  chloride  solution  may  be  used  as  the  diluting 
fluid. 

Fibrinogen  is  perhaps  the  most  important  of  the  protein  constituents 
of  the  plasma.  It  is  also  found  in  lymph  and  chyle  as  well  as  in  certain, 
exudates  and  transudates.  Fibrinogen  possesses  the  general  properties 
of  the  globulins,  but  differs  from  serum  globulin  in  being  precipitated 
upon  half-saturation  with  sodium  chloride.  In  the  process  of  coagula- 
tion of  the  blood  the  fibrinogen  is  transformed  into  fibrin.  This  fibrin  is 
one  of  the  principal  constituents  of  the  ordinary  blood  clot. 

The  nucleoprotein  of  blood  possesses  many  of  the  characteristics  of 
serum  globulin.  In  common  with  this  body  it  is  easily  soluble  in  sodium 
chloride,  and  is  completely  precipitated  from  its  solutions  upon  satura- 
tion with  magnesium  sulphate.  It  is  much  less  soluble  in  dilute  acetic 
acid  than  serum  globulin,  and  its  solutions  coagulate  at  65°-69°C. 

The  body  formerly  called  serum  globulin  is  probably  not  an  indi- 
vidual substance.  Recent  investigations  seem  to  indicate  that  it 
may  be  resolved  into  two  individual  bodies  called  euglobulin  and  pseudo- 
globulin.  The  euglobulin  is  practically  insoluble  in  water  and  may  be 
precipitated  in  the  presence  of  28-36  per  cent  of  saturated  ammonium 
sulphate  solution.  The  pseudo-globulin,  on  the  contrary,  is  soluble  in 
water  and  is  only  precipitated  by  ammonium  sulphate  in  the  presence  of 
from  36  to  44  per  cent  of  saturated  ammonium  sulphate  solution. 

In  common  with  serum  globulin  the  body  known  as  serum  albumin 
seems  also  to  consist  of  more  than  a  single  individual  substance.  The 
so-called  serum  albumin  may  be  separated  into  at  least  two  distinct 
bodies,  one  capable  of  crystallization,  the  other  an  amorphous  body. 
The  solution  of  either  of  these  bodies  in  water  gives  the  ordinary  albu- 
min reactions.  The  coagulation  temperature  of  the  serum  albumin  mix- 
ture as  it  occurs  in  serum  or  plasma  varies  from  70°  to  85°C.  according  to 
the  reaction  of  the  solution  and  its  content  of  inorganic  material. 
Serum  albumin  differs  from  egg  albumin  in  being  more  levorotatory, 
in  being  rendered  less  insoluble  by  alcohol,  and  in  the  fact  that  when 


BLOOD    AND    LYMPH  247 

precipitated  by  hydrochloric  acid  it  is  more  easily  soluble  in  an  excess  of 
the  reagent. 

When  blood  coagulates  and  the  usual  clot  forms,  a  light  yellow  fluid 
exudes.  This  is  blood  serum.  It  differs  from  blood  plasma  in  contain- 
ing a  large  amount  oi  fibrin  ferment,  a  body  of  great  importance  in  the 
coagulation  of  the  blood,  and  also  in  possessing  a  lower  protein  content. 
The  protein  material  present  in  plasma  and  not  found  in  serum  is  the 
fibrinogen  which  is  transformed  into  fibrin  in  the  process  of  coagulation 
and  removed.  The  specific  gravnty  of  the  serum  of  human  blood 
varies  between  1.026  and  1.032.  If  blood  be  drawn  into  a  vessel  and 
allowed  to  remain  without  stirring  or  agitation  of  any  sort  the  major 
portion  of  the  red  corpuscles  will  sink  away  from  the  upper  surface, 
causing  this  portion  of  the  clot  to  assume  a  lighter  color  due  to  the 
predominance  of  leucocytes-  This  light-colored  portion  of  the  clot  is 
called  the  ''  buffy  coat." 

Beside  the  protein  constituents  already  mentioned,  other  bodies 
which  are  found  in  both  the  plasma  and  serum  are  the  following:  Sugar 
{glucose),  uric  acid  (urates),  urea.  fat.  amino-acids.  enzymes,  lecithin, 
creatine,  carbamic  acid,  cholesterol  and  its  esters,  nucleoprotein.  acetone 
bodies,  paralactic  acid,  gases,  ammonia,  coloring-matter  (lutein  or  lipo- 
chrome)  and  mineral  substances.  In  addition  to  the  substances  just 
named  the  blood  doubtless  contains  a  class  of  substances  called  hor- 
manes  of  which  adrenaline  is  the  only  one  thus  far  definitely  identified. 
Some  of  the  pathological  constituents  of  blood  are  proteoses,  biliary  con- 
stituents and  purine  bodies.  In  many  pathological  conditions  certain 
normal  constituents  are  present  in  increased  amount. 

Normal  human  blood  contains  slightly  less  than  o.i  per  cent  of 
glucose  on  the  average.  Strouse^  in  a  very  recent  series  of  tests  places 
the  average  glucose  content  at  0.084  per  cent.  That  the  diet  influences 
the  sugar  content  is  shown  by  the  fact  that  two  and  one-half  to  four 
hours  after  a  meal  the  sugar  content  has  been  found  to  equal  0.18  per 
cent.-  In  case  of  glycosuria  the  blood  sugar  may  increase  (hypergly- 
cemia) to  0.3-1.0  per  cent.  For  the  quantitative  determination  of 
blood  sugar  see  page  279. 

The  determination  of  the  cholesterol  content  of  the  blood  is  assuming 
clinical  importance.  Normal  blood  contains  140-180  mg.  per  100 
grams  of  blood,  or  about  0.15  per  cent.  This  value  has  been  found  to 
be  increased  (hypercholesterolemia)  in  gall  stones,  pregnancy,  nephritis, 
diabetes,  and  arteriosclerosis  and  syphilis.  (See  page  285  for  quantitative 
methods.) 

'  Strouse:  Bull.  Johns  Hop.  Hasp.,  26,  211.  1915. 
^Hirsch:  Zeil.  physiol.  Client.,  93.  355,  1915. 


248  PHYSIOLOGICAL    CHEMISTRY 

Uric  acid  is  present  in  normal  blood  to  the  extent  of  about  1-2  mg. 
per  100  grams  of  blood.  In  gout  this  value  may  be  increased  to  4-6 
mg.  The  quantitative  determination  of  the  uric  acid  content  of  blood 
is  of  importance  as  an  aid  in  differentiating  gout  and  certain  other 
disorders  exhibiting  similar  clinical  symptoms  (for  method  see  page 
274). 

The  non-protein  nitrogen  of  normal  blood  amounts  to  about  25-35 
mg.  per  100  grams  of  blood.  The  urea  forms  about  50  per  cent  of  this, 
creatinine  2  per  cent,  uric  acid  2  per  cent,  ammonia  0.3  per  cent,  and 
amino-acids,  etc.,  about  46  per  cent.  In  nephritis  the  non-protein 
nitrogen  of  the  blood  is  much  increased.  Myers  and  Fine^  report  a 
fatal  case  of  uremia  in  which  the  non-protein  nitrogen  reached  300  mg. 

Amino-acids  are  always  present  in  the  blood.  They  result  for  the 
most  part  from  the  digestion  of  protein  material  in  the  intestine. 

Creatinine  occurs  in  normal  blood  to  the  extent  of  about  1-2  mg. 
per  100  c.c.  of  blood.  In  uremia  the  amount  is  increased.^  Various 
investigators  report  the  values  as  ranging  from  5  to  33  mg.  per 
100  c.c. 

The  creatine  content  of  normal  blood  ranges  from  5  to  10  mg.  per 
100  c.c.  of  blood.  The  creatine  values  have  no  important  pathological 
significance  at  the  present  time. 

The  acetone  (acetone  and  acetoacetic  acid)  content  of  normal  blood 
ranges  from  o  to  i  mg.  per  100  c.c.  of  blood.  In  mild  diabetes  mellitus 
the  value  rises  to  5-12  mg.,  whereas  in  severe  diabetes  mellitus  (coma) 
as  much  as  20-45  ^g-  P^^  1°°  c.c.  of  blood  serum  has  been  found. 

Normal  blood  contains  about  20  per  cent  of  solids  and  3  per  cent  of 
total  nitrogen,  whereas  chlorides  are  present  to  the  extent  of  about  0.65 
per  cent.  In  severe  diabetes  the  chlorides  are  decreased  because  of  the 
accompanying  diuresis. 

AbeP  and  associates  have  devised  a  method  by  which  diffusible 
substances  may  be  removed  from  the  blood  of  a  living  animal.  The 
process  is  termed  vividifusion  and  is  brought  about  by  permitting  the 
blood  from  an  artery  to  flow  through  collodion  tubes  surrounded  by 
physiological  salt  solution.  The  dialyzable  substances,  except  sodium 
chloride,  are  removed  and  the  dialyzed  blood  is  returned  to  the  body  of 
the  animal  by  means  of  a  vein.  The  apparatus  has  been  modified  by 
McGuigan  and  von  Hess."* 

1  Myers  and  Fine:  Chemical  Composition  of  the  Blood  in  Health  and  Disease,  1915. 
^  Folin  and  Denis;  Jour.  Biol.  Chcm.,  17,  487,  1914. 
Myers  and  Fine:  Jour.  Biol.  Chcm.,  20,  391,  191 5. 
3  Abel,  Rowntree  and  Turner:   Transactions  of  the  Ass'n  of  American  Physicians,  1913; 
also  Jour,  of  P harm,  and  Exp.  Therap.,  5,  275,  1914. 

*  McGuigan  and  Von  Hess:  Jour.  Pharm.  and  E.xp.  Therap.,  vol.  6,  1914'. 


PLATE  IV. 


Normal  Erythrocytes  and  Leucocytes. 


BLOOD    AND    LYMPH  249 

In  the  application  of  blood-letting  or  venesection  it  has  been  cus- 
tomary to  discard  both  the  corpuscles  and  plasma  of  the  withdrawn 
blood.  AbeP  and  associates  have  found  it  possible  to  separate  the 
corpuscles  from  the  removed  blood  by  centrifugation  and  to  return 
them  to  the  body  suspended  in  Locke's  solution.  They  name  the  pro- 
cedure plasma phcBresis.  By  this  means  blood-letting  can  be  carried 
out  repeatedly  during  a  short  interval  of  time  without  endangering  the 
life  of  the  animal. 

There  has  been  considerable  controversy  regarding  the  form  of  the 
erythrocytes  or  red  blood  corpuscles  of  human  blood.  It  is  claimed  by 
some  investigators  that  the  cells  are  hell-shaped  or  cup-shaped.  As  the 
erythrocytes  occur  normally  in  the  circulation,  however,  they  are  prob- 
ably thin,  non-nucleated,  biconcave  discs.- 

The  blood  of  most  mammals  contains  erythrocytes  similar  in  form 
to  those  of  human  blood.  In  the  blood  of  birds,  fishes,  amphibians  and 
reptiles  the  erythrocytes  are  ordinarily  more  or  less  elliptical,  biconvex 
and  possess  a  nucleus.  The  erythrocytes  vary  in  size  with  the  different 
animals.  The  average  diameter  of  the  erythrocytes  of  blood  from 
various  species  is  given  in  the  following  table  :^ 

Elephant .  -V,  ,f  of  an  inch. 

Guinea-pig .,-2^3-  of  an  inch. 

Man jji.  jj  of  an  inch. 

Monkey ,~'j^  of  an  inch. 

Dog .,,ig  j^  of  an  inch. 

Rat. ._ j^ij .,  of  an  inch. 

Rabbit ^gijj  of  an  inch. 

Mouse ^>.V3  of  an  inch. 

Lion 5^-ji^^  of  an  inch. 

Ox :i2  r?  of  an  inch. 

Horse -j-^^  of  an  inch. 

Pig 3^^5  of  an  inch. 

Cat -i^^  of  an  inch. 

Sheep ;,  j,Jy;  of  an  inch. 

Goat T,-^^,  of  an  inch. 

Musk-deer i ,  j  i  ^  of  an  inch. 

The  erythrocytes,  from  whatever  source  obtained,  consist  essentially 
of  two  parts,  the  stroma  or  protoplasmic  tissue  and  its  enclosed  pigment, 
hemoglobin.  For  human  blood  the  number  of  erythrocytes  present  in 
the  fluid  as  obtained  from  well-developed  males  in  good  physical  condi- 
tion is  about  5,500,000  per  cubic  millimeter."*  The  normal  content  of 
the  blood  of  adult  females  is  from  4,000,000  to  4,500,000  per  cubic 

^Abel,  Rowntree,  Turner,  Marshall  and  Lamson  (see  Abel's  Mellon  Lecture,"  1915): 
also  Abel:  Science,  42,  135,  1915. 

*  When  examined  singly  under  the  microscope,  they  possess  a  pale  greenish-yellow 
color  (see  Plate  IV,  opposite),  whereas  when  grouped  in  large  masses  a  reddish  tint  is 
noted. 

'  Wormley's  Micro-Chemistry  of  Poisons,  second  edition,  p.  733. 

*  This  statement  is  based  upon  observations  made  upon  the  blood  of  athletes  in  training. 
See  Hawk:  Amer.  Jour.  Physiol., 10,  384,  1904.  It  is  generally  stated  in  text-books  that 
the  blood  of  males  contains  about  5,000,000,  per  cubic  millimeter. 


250  PHYSIOLOGICAL    CHEMISTRY 

millimeter.  The  number  of  erythrocytes  varies  greatly  under  different 
conditions.  For  instance,  the  number  may  be  increased  after  the  trans- 
fusion of  blood  of  the  same  species  of  animal;  by  residing  in  a  high 
altitude;  or  as  a  result  of  strenuous  physical  exercise  continued  over  a 
short  period  of  time.  An  increase  is  also  noted  in  starvation;  after 
partaking  of  food;  after  cold  or  hot  baths;  after  massage,  as  well  as 
after  the  administration  of  certain  drugs  and  accompanying  certain 
diseases,  such  as  cholera,  diarrhea,  dysentery  and  yellow  atrophy  of  the 
liver.  A  decrease  in  the  number  occurs  in  the  different  forms  of  anemia. 
The  number  has  been  known  to  increase  to  7,040,000  per  cubic  milli- 
meter as  a  result  of  physical  exercise,  while  11,000,000  per  cubic  milli- 
meter have  been  noted  in  cases  of  polycythemia  and  increases  nearly  as 
great  in  cyanosis.  The  number  has  been  known  to  decrease  to  500,000 
per  cubic  millimeter  or  lower  in  pernicious  anemia. 

Erythrocytes  possess  the  property,  when  properly  treated,  of 
"clumping"  together  in  masses  and  precipitating,  producing  so-called 
agglutination.  Cells  other  than  erythrocytes  {e.g.,  bacteria)  possess 
this  property.  When  spoken  of  in  connection  with  the  blood  such 
action  is  termed  hemagglutination.  A  substance  which  will  bring  about 
hemagglutination  is  said  to  contain  hemagglutinins.  These  hem- 
agglutinins are  particularly  abundant  in  the  vegetable  kingdom.^  For 
a  demonstration  of  hemagglutination  see  page  261. 

Oxyhemoglobin,  the  coloring  matter  of  the  blood,  is  a  conjugated 
protein.  Through  treatment  with  hydrochloric  acid  it  may  be  split  into 
a  protein  body  called  globin,  and  hemochromogen,  an  iron-containing 
pigment.  The  latter  body  is  rapidly  transformed  into  hematin  in  the 
presence  of  oxygen,  and  this  in  turn  gives  place  to  hematin-hydrochlo- 
ride  or  hemin  (Figs.  78  and  79,  page  265).  The  pigment  of  arterial  blood 
is  for  the  most  part  loosely  combined  with  oxygen  and  is  termed  oxy- 
hemoglobin,  whereas  the  pigment  of  venous  blood  is  principally  hemo- 
globin (so-called  reduced  hemoglobin) .  Oxyhemoglobin  is  the  oxygen 
carrier  of  the  body  and  belongs  to  the  class  of  bodies  known  as  respira- 
tory pigments.  It  is  held  within  the  stroma  of  the  erythrocyte.  The  re- 
duction of  oxyhemoglobin  to  form  hemoglobin  (so-called  reduced  hemo- 
globin) occurs  in  the  capillaries.  Oxyhemoglobin  may  be  crystallized 
and  a  specific  form  of  crystal  obtained  from  the  blood  of  each  individual 
species  (see  Figs.  70  to  76,  pages  251  to  254).  This  fact  seems  to  indi- 
cate that  there  are  many  varieties  of  oxyhemoglobin.  The  interesting 
findings  of  Reichert  and  Brown  are  of  great  value  in  this  connection. 
These  investigators  prepared  oxyhemoglobin  crystals  from  the  blood 

^Mendel:  Archivio  di  fisiologia,  7,  168,  1909;  also  Schneider:  Journal  of  Biological 
Chem.,  II,  47,  1912. 


BLOOD    AND    LYMPH 


2qi 


Fig.  70. — Oxyhemoglobin  Crystals  from  Blood  of  the  Guinea-pig. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


^x'^ 


\ 


<  h 


1 


^-/ 


4k 


Fig.  71. — Oxyhemoglobin  Crystals  from  Blood  of  the  Rat. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsvlvania. 


252 


PHYSIOLOGICAL   CHEMISTRY 


Fig.  72. — Oxyhemoglobin  Crystals  from  Blood  of  the  Horse. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


# 


Fig.  73. — Oxyhemoglobin  Crystals  from  Blood  of  the  Squirrel. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


BLOOD   AND    LYMPH 


253 


Fig.  74. — OxYHEiioGLOBix  Crystals  from  Blood  of  the  Dog. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


Fig.  75. — Oxyhemoglobin'  Crystals  from  Blood  or  the  C.\t. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsvlvania. 


254 


PHYSIOLOGICAL    CHEMISTRY 


of  over  one  hundred  species  of  animal  and  subsequently  studied  the 
characteristics  of  the  crystals  very  minutely  from  the  standpoint  of 
crystallography.  Their  findings  may  prove  of  importance  from  the 
standpoint  of  heredity  and  the  origin  of  species. 


Fig.  76. — Oxyhemoglobin  Crystals  from  Blood  of  the  Necturus. 
Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania.  1 


HEMOGLOBIN 


Carbon  monoxide  hemoglobin 


Acid  hematin  (+  globin) 


■fi" 

0 

•X3 

CO 

ei 

^— s 

■^ 

■^ 

'p 

X 

0^ 

^ 

v^ 


Oxyhemoglobin 


Hemin  ^/)J 

(Hematin  hydrochloride)  N  .  _      . 

Acid  hematoporph3rrin  (+  iron) 


Hemochromogen 
Methemoglobin 

3  . 


ra   t-r' 


\^e 


Alkaline  hematin  (+  globin) 


Hemin 

(Hematin  hydrochloride) 


Alkaline  hematoporph3^in 

*  The  micro-photographs  of  oxyhemoglobin  (see  pages  251-254)  and  hemin  (see  page 
(265)  are  reproduced  through  the  courtesj^  of  Professors  E.  T.  Reichert  and  Amos  P.  Brown, 
of  the  University  of  Pennsylvania,  who  have  investigated  the  crystalline  forms  of 
biochemic  substances. 


BLOOD    AND    LYMPH  255 

The  following  bodies  may  be  derived  from  hemoglobin,  and  each 
possesses  a  specific  spectrum  which  serves  as  an  aid  in  its  detection 
and  identification:  Oxyhemoglobin,  methemoglobin,  carbon-monox- 
ide hemoglobin,  nitric-oxide  hemoglobin,  hemochromogen,  hematin, 
acid-hematin,  alkali-hematin  and  hematoporphyrin  (see  Absorption 
Spectra,  Plates  I  and  II). 

The  relationship  between  hemoglobin  and  its  derivatives  may  be 
represented  by  the  scheme  shown  on  page  254. 

The  chemical  transformations  which  occur  in  the  blood  duringrespira- 
tion  are  complicated  and  of  great  importance.  In  brief  the  exchange  of 
oxygen  and  carbon  dioxide  may  be  described  as  follows:  Oxygen  from 
the  air  passes  through  the  lungs  into  the  blood  where  it  is  appropriated 
for  the  most  part  by  the  red  blood  corpuscles.  The  hemoglobin  of 
these  corpuscles  possesses  the  property  of  uniting  with  oxygen,  forming 
oxyhemoglobin.  This  oxyhemoglobin  possesses  a  red  color  and  imparts 
to  the  arterial  blood  its  bright  appearance.  The  oxygen  is  thus  borne 
by  these  blood  cells  in  the  circulating  blood  to  all  parts  of  the  body. 
As  the  blood  passes  through  the  capillaries  it  gives  up  the  major  part  of 
its  oxygen  which  is  used  by  the  tissues  in  their  varied  activities.  As  the 
blood  loses  its  oxygen  it  becomes  darker  in  color  due  to  the  fact  that 
the  oxyhemoglobin  has  been  transformed  into  hemoglobin  (or  reduced 
hemoglobin).  At  the  same  time  in  the  tissue  capillaries  the  blood  takes 
up  excretory  products  from  the  tissues,  the  chief  of  which  is  carbon 
dioxide.  This  carbon  dioxide  is  present  in  the  blood  mainly  as  sodium 
carbonate  and  sodium  acid  carbonate;  a  small  amount  is  probably  com- 
bined with  the  proteins  of  the  plasma.  We  now  have  so-called  venous 
blood.  This  is,  in  turn,  carried  to  the  lungs  where  the  carbon  dioxide  is 
exchanged  for  oxygen  and  the  cycle  is  repeated. 

The  white  corpuscles  (or  leucocytes)  of  human  blood  differ  from 
the  red  corpuscles  (or  erythrocytes)  in  many  particulars,  such  as  being 
somewhat  larger  in  size,  in  containing  at  least  a  single  nucleus  and  in 
possessing  ameboid  movement  (see  Plate  IV,  opposite  page  249). 
They  are  typical  animal  cells  and  therefore  contain  the  following  bodies 
which  are  customarily  present  in  such  cells:  Proteins,  fats,  glycogen, 
purine  bodies,  enzymes,  phosphatides,  lecithin,  cholesterol,  inorganic  salts 
and  water.  Compound  proteins  make  up  the  chief  part  of  the  protein 
quota  of  leucocytes,  the  nucleoproteins  predominating.  Of  the  en- 
zymes present  the  proteolytic  are  the  most  important.  It  is  claimed^ 
that  there  are  two  proteolytic  enzymes  in  leucocytes,  one  active  in 
alkaline  solution  and  present  in  the  polynuclear  cells,-  and  the  other 

*Opie:  Jour,  of  Ex  peri  menial  Med.,  8;  Opie  and  Barker:  Ibid.,  9. 
2  For  discussion  of  different  types  of  leucocytes  see  "Da  Costa's  Clinical  Hematology" 
or  some  similar  volume. 


256  PHYSIOLOGICAL    CHEMISTRY 

active  in  acid  medium  and  present  in  mononuclear  cells.  It  is  claimed 
that  the  granular  leucocytes  originate  in  the  bone  marrow,  whereas 
the  non-granular  leucocytes  (lymphocytes)  have  a  lymphatic  origin 
(lymph  glands  or  lymphoid  tissue) ;  this  matter  of  origin  is  uncertain. 
The  normal  number  of  leucocytes  in  human  blood  varies  between  5000 
and  10,000  per  cubic  millimeter.  The  ratio  between  the  leucocytes  and 
erythrocytes  is  about  1:350-500.  A  leucocytosis  is  said  to  exist  when 
the  number  of  leucocytoses  is  increased  for  any  reason.  Leucocytoses 
may  be  divided  into  two  general  classes,  the  physiological  *and  the 
pathological.  Under  the  physiological  form  would  be  classed  those 
leucocytoses  accompanying  pregnancy,  parturition  and  digestion,  as  well 
as  those  due  to  mechanical  and  thermal  influences.  The  leucocytoses 
spoken  of  as  pathological  are  the  inflammatory,  infectious,  post-hemor- 
rhagic,  toxic  and  experimental  forms,  as  well  as  the  type  of  leucocytosis 
which  accompanies  malignant  disease. 

The  blood  plates  (platelets  or  plaques)  are  round  or  oval  colorless 
discs  which  possess  a  diameter  about  one-third  as  great  as  that  of  the 
erythrocytes.  Upon  treatment  with  certain  reagents,  e.g.,  artificial 
gastric  juice,  they  may  be  separated  into  a  homogeneous,  non-refractive 
portion  and  a  granular,  refractive  portion.  The  blood  plates  are 
associated  with  the  coagulation  of  the  blood.  This  relationship  is  not 
completely  understood  at  present. 

The  hemoconein  or  so-called  "blood  dust"  is  made  up  of  round 
granules  which  usually  have  a  diameter  somewhat  less  than  i  micron. 
The  serum  of  normal  as  well  as  of  pathological  blood  contains  these 
granules.  They  were  first  described  by  Miiller  to  whom  they  appeared 
as  highly  refractile  granules  possessed  of  Brownian  movement.  The 
"blood  dust"  is  apparently  not  concerned  with  the  coagulation  of  the 
blood.  The  granules  are  insoluble  in  alcohol,  ether  and  acetic  acid 
and  are  not  blackened  by  osmic  acid.  According  to  Miiller,  the  gran- 
ules making  up  the  so-called  "blood  dust"  constitute  a  new  organized 
constituent  of  the  blood,  whereas  other  investigators  believe  them  to  be 
merely  free  granules  from  certain  of  the  forms  of  leucocytes.  They 
appear  to  possess  no  clinical  significance. 

The  processes  involved  in  the  coagulation  of  the  blood  are  not  fully 
understood.  Several  theories  have  been  advanced  and  each  has  its 
adherents.  The  theory  which  appears  to  be  fully  as  firmly  founded 
upon  experimental  evidence  as  any  is  the  following:  Blood  contains  a 
zymogen  called  prothrombin  which  combines  with  the  calcium  salts 
present  to  form  an  enzyme  known  as  thrombin  or  fibrin-ferment.  When 
freshly  drawn  blood  comes  into  contact  with  the  air  the  fibrin-ferment 
at  once  acts  upon  the  fibrinogen  present  and  gives  rise  to  the  formation 


BLOOD    AND    LYMPH  257 

oi  fibrin.  This  fibrin  forms  in  shreds  throughout  the  blood  mass  and, 
holding  the  form  elements  of  the  blood  within  its  meshes,  serves  to 
produce  the  typical  blood  clot.  The  fibrin  shreds  gradually  contract, 
the  whole  clot  assumes  a  jelly-like  appearance  and  the  yellowish  serum 
exudes.  If,  immediately  upon  the  withdrawal  of  blood  from  the  body, 
the  fluid  be  rapidly  stirred  or  thoroughly  "whipped"  with  a  bundle  of 
coarse  strings,  twigs  or  a  specially  constructed  beater,  the  fibrin  shreds 
will  not  form  in  a  network  throughout  the  blood  mass  but  instead  will 
cling  to  the  device  used  in  beating.  In  this  way  the  fibrin  may  be 
removed  and  the  remaining  fluid  is  termed  defibrinated  blood.  The 
above  theory  of  the  coagulation  of  the  blood  may  be  stated  briefly  as 
follows : 

I.  Prothrombin  +  Calcium  Salts  =  Thrombin  (or  Fibrin-ferment). 

II.  Thrombin  (or  Fibrin-ferment)  -f  Fibrinogen  =  Fibrin. 
HowelP  has  suggested  an  ingenious  modification  of  the  above  theory. 

He  says:  "In  the  circulating  blood  we  find  as  constant  constituents 
fibrinogen,  prothrombin,  calcium  salts  and  antithrombin.  The  last-named 
substance  holds  the  prothrombin  in  combination  and  thus  prevents  its 
conversion  or  activation  to  thrombin.  When  the  blood  is  shed,  the 
disintegration  of  the  corpuscles  (platelets)  furnishes  material  (throm- 
boplastin) which  combines  with  the  antithrombin  and  liberates  the 
prothrombin;  the  latter  is  then  activated  by  the  calcium  and  acts  on 
the  fibrinogen.  According  to  this  view  the  actual  process  of  coagula- 
tion involves  only  three  factors,  fibrinogen,  prothrombin  and  calcium. 
These  three  factors  exist  normally  in  the  circulating  blood  but  are 
prevented  from  reacting  by  the  presence  of  antithrombin." 

The  question  as  to  whether  menstrual  blood  coagulates  has  caused 
much  discussion.  The  most  recent  investigations  seem  to  show  that  it 
does  not  coagulate  because  of  the  removal  of  fibrin-ferment  and  fibrinogen 
from  such  blood  by  the  endometrium  or  lining  membrane  of  the  uterus.^ 

Among  the  medico-legal  tests  for  blood  are  the  following:  (i) 
Microscopical  identification  of  the  erythrocytes,  (2)  spectroscopic  iden- 
tification of  blood  solutions,  (3)  the  guaiac  test,  (4)  the  benzidine  reac- 
tion, (5)  preparation  of  hemin  crystals.  Of  these  five  tests  the  last 
two  named  are  generally  considered  to  be  the  most  satisfactory.  Tliey 
give  equally  reliable  results  with  fresh  blood  and  ^^ith  blood  from  clots 
or  stains  of  long  standing,  provided  the  latter  have  not  been  exposed  to 
a  high  temperature  or  to  the  rays  of  the  sun  for  a  long  period.  The 
technic  of  the  tests  is  simple  and  the  formation  of  the  dark  brown  or 
chocolate-colored  crystals  of  hemin  or  the  production  of  the  green  or 

'  Howell:  American  Journal  of  Physiology,  29,  187,  191 1. 
*  Bell:  Jour.  Path,  and  Bad.,  18,  No.  4,  1914. 

17 


258  PHYSIOLOGICAL   CHEMISTRY 

blue  color  with  benzidine  is  indisputable  proof  of  the  presence  of  blood 
in  the  fluid,  clot  or  stain  examined.  The  weak  point  of  the  tests, 
medico-legally,  lies  in  the  fact  that  they  do  not  differentiate  between 
human  blood  and  that  of  certain  other  species  of  animal. 

The  guaiac  test  (see  page  262),  although  generally  considered  less 
accurate  than  the  hemin  test,  is  held  by  some  to  be  a  more  delicate  test 
than  the  hemin  test  if  properly  performed.  One  of  the  most  common 
mistakes  in  the  manipulation  of  this  test  is  the  use  of  a  guaiac  solution 
which  is  too  concentrated  and  which,  when  brought  into  contact  with 
the  aqueous  blood  solution,  causes  the  separation  of  a  voluminous 
precipitate  of  a  resinous  material  which  may  obscure  the  blue  colora- 
tion; this  is  particularly  true  of  the  test  when  used  for  the  examination 
of  blood  stains.  A  solution  of  guaiac  made  by  dissolving  i  gram  of  the 
resin  in  60  c.c.  of  95  per  cent  alcohol  is  very  satisfactory  for  general  use. 
The  test  is  frequently  objected  to  upon  the  ground  that  various  other 
substances,  e.g.,  milk,  pus,  saliva,  etc.,  respond  to  the  test  and  that  it 
cannot  therefore  be  considered  a  specific  test  for  blood  and  is  of  value 
only  in  a  negative  sense.  We  have  demonstrated  to  our  own  satisfac- 
tion, however,  that  many  samples  of  milk  give  the  blue  color  upon  the 
addition  of  an  alcoholic  solution  of  guaiac  resin  without  the  addition  of 
hydrogen  peroxide  or  old  turpentine.  It  has  also  been  shown^  that 
those  milks  which  respond  positively,  fail  to  do  so  after  boiling.  In  the 
case  of  blood  the  test  is  positive  both  before  and  after  boiling  the  blood 
for  15-20  seconds.  Pus  does  not  respond  after  boiling.  Old,  partly 
putrilied  pus  gives  the  test  even  without  the  addition  of  hydrogen 
peroxide  or  old  turpentine,  whereas  fresh  pus  responds  upon  the  addi- 
tion of  hydrogen  peroxide.  Saliva  gives  a  positive  reaction  only  in 
case  blood  or  pus  is  present.  Certain  plant  extracts  give  the  test  before 
but  not  after  boiling  for  15-20  seconds.  Buckmaster  has  advocated 
the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an  alcoholic 
solution  of  guaiac  resin.  He  claims  that  he  was  able  to  produce  the 
blue  color  upon  the  addition  of  the  guaiaconic  acid  to  milk  only  when 
the  sample  of  milk  tested  was  brought  from  the  country  in  sterile  bottles, 
and  further,  that  no  sample  of  London  milk  which  he  examined  responded 
to  the  test.  In  the  application  of  the  guaiac  test  to  the  detection  of 
blood,  he  states  that  he  was  able  to  detect  laked  blood  when  present  in 
the  ratio  i  :  5,000,000  and  unlaked  blood  when  present  in  the  ratio 
I  :  1,000,000.  This  author  considers  the  guaiac  test  to  be  far  more 
trustworthy  than  is  generally  believed. 

Up  to  within  recent  times  it  has  been  impossible  to  make  an  absolute 
differentiation  of  human  blood.     The  so-called  "biological"  blood  test 

^Leary:  Private  communication. 


BLOOD    AND    LYilPH  259 

has,  however,  made  such  a  differentiation  possible.  ITiis  test,  known  as 
the  Bordet  reaction,  is  founded  upon  the  fact  that  the  blood  serum  of  an 
animal  into  which  has  been  injected  the  blood  of  another  animal  of 
different  species  develops  the  property  of  agglutinating  and  dissoK-ing 
erythrocytes '  5/w//ar  to  those  injected,  but  exerts  this  influence  upon 
the  blood  from  no  other  species.  The  antiserum  used  in  this  test  is 
prepared  by  injecting  rabbits  with  5-10  c.c.  of  human  defibrinated 
blood,  at  intervals  of  about  four  days,  until  a  total  of  between  50  and  80 
c.c.  has  been  injected.  After  a  lapse  of  one  or  two- weeks  the  animal  is 
bled,  the  serum  collected,  placed  in  sterile  tubes  and  preserved  for  use  as 
needed.  In  examining  any  specific  solution  for  human  blood  it  is  simply 
necessary  to  combine  the  antiserum  and  the  solution  under  examination 
in  the  proportion  of  1:100  and  place  the  mixture  at  37°C.  If  human 
blood  is  present  in  the  solution  a  turbidity  wdll  be  noted  and  this  will 
change  within  three  hours  to  a  distinctly  flocculent  precipitate.  This 
antiserum  will  react  thus  with  no  other  known  substance. 

Lymph  may  be  considered  as  the  "middle  man"  in  the  transactions 
between  blood  and  tissues.  It  is  the  medium  by  which  the  nutritive 
material  and  oxygen  transported  by  the  blood  for  the  tissues  is  brought 
into  intimate  contact  with  those  tissues  and  thus  utilized.  In  the 
further  fulfillment  of  its  function,  the  lymph  bears  from  the  tissues 
water,  salts  and  the  products  of  the  acti\dty  and  catabolism  of  the 
tissues  and  passes  these  into  the  blood.  Lymph,  therefore,  exercises 
the  function  of  a  "go-between"  for  blood  and  tissues.  It  bathes  every 
active  tissue  of  the  animal  body,  and  is  believed  to  have  its  origin  partly 
in  the  blood  and  partly  in  the  tissues. 

In  chemical  characteristics,  lymph  resembles  blood  plasma.  In  fact, 
it  has  been  termed  "blood  without  its  red  corpuscles."  L>Tnph  from 
the  thoracic  duct  of  a  fasting  animal  or  from  a  large  lymphatic  vessel  of 
a  well-nourished  animal  is  of  a  variable  color  (colorless,  yellowish  or 
slightly  reddish)  and  alkaline  in  reaction  to  litmus.  It  contains 
fibrinogen,  fibrin-ferment  and  leucocytes  and  coagulates  slowly,  the  clot 
being  less  firm  and  bulky  than  the  blood  clot.  Serum  albumin  and 
serum  globulin  are  both  present  in  lymph,  the  albumin  predominating 
in  a  ratio  of  about  3  or  4:1.  The  principal  inorganic  salts  are  sodium 
salts  (chloride  and  carbonate),  whereas  the  phosphates  of  potassium, 
calcium,  magnesium  and  iron  are  present  in  smaller  amount. 

Substances  which  stimulate  the  flow  of  lymph  are  termed  lympha- 
gogiies.  Such  substances  as  sugar,  urea,  certain  salts  (especially 
sodium  chloride),  peptone,  egg  albumin,  extracts  of  dogs'  liver  and 
intestine,  crab  muscles  and  blood  leeches  are  included  in  this  class. 

In  a  fasting  animal,  the  lymph  coming  from  the  intestine  is  a  clear, 


26o  PHYSIOLOGICAL    CHEMISTRY 

transparent  fluid  possessing  the  characteristics  already  outlined.  After 
a  meal  containing  fat  has  been  ingested,  this  intestinal  lymph  is  white 
or  "  milky."  This  is  termed  chyle  and  is  essentially  lymph  possessing  an 
abnormally  high  (5-15  per  cent)  content  of  emulsified  fat.  This  chyle 
is  absorbed  by  the  lacteals  of  the  intestine  and  transported  to  the  lower 
portion  of  the  thoracic  duct.  Apart  from  the  fat  value,  the  composition 
of  lymph  and  chyle  are  similar. 

Experiments  on  Blood 

I.  Defibrinated  Ox-blood 

1.  Reaction. — Moisten  red  and  blue  litmus  papers  with  10  per  cent  sodium 
chloride  solution  and  test  the  reaction  of  the  defibrinated  blood.  Test  by  Congo 
red  paper  also. 

2.  Microscopical  Examination. — ^Examine  a  drop  of  defibrinated  blood  under 
the  microscope.  Compare  the  objects  you  observe  with  Plate  IV,  opposite  page 
249.    Repeat  the  test  with  a  drop  of  yoxxr  own  blood. 

3.  Specific  Gravity. — ^Determine  the  specific  gravity  of  defibrinated  blood 
by  means  of  an  ordinary  specific  gravity  spindle.  Compare  this  result  with 
the  specific  gravity  as  determined  by  Hammerschlag's  method  in  the  next 
experiment. 

4.  Specific  Gravity  by  Hammerschlag's  Method. — Fill  an  ordinary  urinom- 
eter  cylinder  about  one-half  full  of  a  mixture  of  chloroform  and  benzene,  having 
a  specific  gravity  of  approximately  1.050.  Into  this  mixture  allow  a  drop  of  the 
blood  under  examination  to  fall  from  a  pipette  or  directly  from  the  finger  in  case 
fresh  blood  is  being  examined.  Care  must  be  taken  not  to  use  too  large  a  drop 
of  blood  and  to  keep  the  drop  from  coming  into  contact  with  the  walls  of  the  cyl- 
inder. If  the  blood  drop  sinks  to  the  bottom  of  the  vessel,  thus  showing  it  to  be 
of  higher  specific  gravity  than  the  surroimding  fluid,  add  chloroform  until  the 
blood  drop  remains  suspended  in  the  mixture.  Stir  carefully  with  a  glass  rod 
after  adding  the  chloroform.  If  the  blood  drop  rises  to  the  surface  upon  being 
introduced  into  the  mixture,  thus  showing  it  to  be  of  lower  specific  gravity  than  the 
surrounding  fluid,  add  benzene  until  the  blood  drop  remains  suspended  in  the 
mixtiu-e.  Stir  with  a  glass  rod  after  the  benzene  is  added.  After  the  blood 
drop  has  been  brought  to  a  suspended  position  in  the  mixture  by  means  of  one  or 
more  additions  of  chloroform  and  benzene  this  final  mixture  should  be  filtered 
through  muslin  and  its  specific  gravity  accurately  determined.  What  is  the 
specific  gravity  of  the  blood  under  examination? 

5.  Tests  for  Various  Constituents. — ^Place  10  c.c.  of  defibrinated  blood  in  an 
evaporating  dish,  dilute  with  100  c.c.  of  water  and  heat  to  boiling.  Is  there  any 
coagulation,  and  if  so  what  bodies  form  the  coagxilum?  At  the  boiUng-point 
acidulate  slightly  with  dilute  acetic  acid.  Filter.  The  filtrate  should  be  clear 
and  the  coagulum  dark  brown.  Reserve  this  coagulum.  What  body  gives  the 
coagulum  this  color?  Evaporate  the  filtrate  to  about  25  c.c,  filtering  off  any 
precipitate  which  may  form  in  the  process.  Make  the  following  tests  upon  the 
filtrate : 

(a)  Fehling's  Test. — To  5  c.c.  of  theneutraUzed  filtrate  add  5  drops  of  Fehhng's 
solution  and  boil  one  minute. 


BLOOD    AND    LYMPH  26 I 

(b)  Chlorides. — ^To  a  small  amount  of  the  filtrate  in  a  test-tube  add  a  few- 
drops  of  nitric  acid  and  a  Uttle  silver  nitrate.  In  the  presence  of  chloride,  a  white 
precipitate  of  silver  chloride  will  form. 

(c)  Phosphates. — Test  for  phosphates  by  nitric  acid  and  molybdate  solution 
according  to  directions  given  on  page  59. 

(d)  Proteose  and  Peptone. — Test  a  small  amount  of  the  solution  for  pro- 
teose and  peptone  by  saturating  with  ammonium  sulphate  according  to  directions 
given  on  page  120. 

(e)  Crystallization  of  Sodium  Chloride. — Place  the  remainder  of  the  filtrate 
in  a  watch  glass  and  evaporate  it  on  a  water-bath.  Examine  the  crystals  under 
the  microscope  and  compare  them  with  those  in  Fig.  80,  page  267. 

6.  Test  for  Iron. — Incinerate  a  small  portion  of  the  coagulum  from  the  last 
experiment  (5)  in  a  porcelain  crucible.  Cool,  dissolve  the  residue  in  dilute  hy- 
drochloric acid  and  test  for  iron  by  potassiimi  ferrocyanide  or  ammonium  thio- 
cyanate.     Which  of  the  constituents  of  the  blood  contains  the  iron? 


Fig.  77. — Efffxt  of  Water  on  Erythrocytes. 

7.  Hemolysis  ("Laky  Blood"). — Note  the  opacity  of  ordinary  defibrinated 
blood.  Place  a  few  cubic  centimeters  of  this  blood  in  a  test-tube  and  add  water, 
a  httle  at  a  time,  until  the  blood  is  rendered  transparent.  Hemolysis  has  taken 
place.  How  does  the  water  act  in  causing  this  transparency?  Examine  a  drop 
of  hemolyzed  blood  under  the  microscope.  How  does  its  microscopical  appear- 
ance differ  from  that  of  unaltered  blood?  What  other  agents  may  be  used  to 
bring    about    hemolysis? 

8.  Osmotic  Pressure. — Place  a  few  cubic  centimeters  of  blood  in  each  of 
three  test-tubes.  Hemolyze  the  blood  in  the  first  tube  according  to  directions 
given  in  the  last  experiment  (7) :  add  an  equal  volimie  of  isotonic  (0.9  per  cent) 
sodium  chloride  to  the  blood  in  the  second  tube,  and  an  equal  volume  of  10  per 
cent  sodium  chloride  to  the  blood  in  the  third  tube.  Mix  thoroughly  by  shaking 
and  after  a  few  moments  examine  a  drop  from  each  of  the  three  tubes  under  the 
microscope  (see  Figs.  77  and  150,  page  473).  What  do  you  find  and  what  is 
your  explanation  from  the  standpoint  of  osmotic  pressure? 


262  PHYSIOLOGICAL   CHEMISTRY 

9.  Hemagglutination. — The  common  garden  bean,  such  as  the 
Scarlet  Runner/  contains  a  protein  substance  which  exhibits  the 
interesting  property  of  causing  a  clumping  or  agglutination  of  red 
blood  corpuscles.^ 

Dilute  defibrinated  blood^  ten  times  with  physiological  sodium  chloride  solu- 
tion (0.9  per  cent)  and  place  i  c.c.  in  each  of  three  small  test-tubes. 

Grind  three  beans  in  a  coffee  mill,  or  with  mortar  and  pestle  to  a  fine  meal 
and  extract  for  a  few  minutes  with  0.9  per  cent  sodium  chloride  solution.  Filter 
and  add  0.05  c.c.  (about  2-3  drops)  of  the  filtered  extract  to  the  first  of  the  blood 
tubes ;  o.oi  c.c.  to  the  second ;  and  0.05  of  0.9  per  cent  sodium  chloride  to  the  third. 

Invert  each  tube  to  mix  the  contents  thoroughly,  and  note  the  rapid  agglutina- 
tion and  precipitation  of  the  blood  corpuscles  in  the  first  tube,  a  less  rapid  aggluti- 
nation in  the  second,  while  the  third  or  control  tube  remains  unaltered.  In 
one-half  hour  the  corpuscles  in  the  first  tube  often  are  packed  soUd  and  one  is 
able  to  pour  off  perfectly  clear  senmi. 

If  the  remainder  of  the  bean  extract  is  boiled  for  a  few  minutes,  the  coagulmn 
filtered  out  and  0.05  c.c.  of  the  filtrate  added  to  the  control  tube,  still  no  agglutina- 
tion occurs,  indicating  that  the  hemagglutinin  has  been  destroyed  or  removed 
by  the  boihng. 

10.  Diffusion  of  Hemoglobin. — ^Prepare  some  hemolyzed  ("laky")  blood, 
thus  hberating  the  hemoglobin  from  the  erythrocytes.  Test  the  diffusion  of  the 
hemoglobin  by  preparing  a  dialyzer  Uke  one  of  the  models  shown  in  Fig.  2,  page 
24.     How  does  hemoglobin  differ  from  other  well-known  crystalUzable  bodies? 

11.  Guaiac  Test. — To  5  c.c.  of  water  in  a  test-tube  add  2  drops  of  blood. 
By  means  of  a  pipette  drop  an  alcohoUc  solution  of  guaiac  'strength  about  i  :  60)* 
into  the  resulting  mixture  xmtil  a  turbidity  is  observed  and  add  old  turpentine 
or  hydrogen  peroxide,  drop  by  drop,  until  a  blue  color  is  obtained. 

In  the  detection  of  small  amounts  of  blood  the  quantity  of  guaiac 
used  should  also  be  decreased.  Do  any  other  substances  respond  in  a 
similar  manner  to  this  test?  Is  a  positive  guaiac  test  a  sure  indication 
of  the  presence  of  blood?     (See  discussion  on  page  258.) 

12.  Schumm's  Modification  of  the  Guaiac  Test. — To  about  5  c.c.  of  the 
solution  under  examination^  in  a  test-tube  add  about  10  drops  of  freshly  prepared 
alcoholic  solution  of  guaiac.  Agitate  the  tube  gently,  add  about  20  drops  of  old 
turpentine,  subject  the  tube  to  a  thorough  shaking  and  permit  it  to  stand  for  about 
two  to  three  minutes.  A  blue  color  indicates  the  presence  of  blood  in  the  solution 
under  examination.     In  case  there  is  insufficient  blood  to  yield  a  blue  color  under 

^  The  Scarlet  Runner  is  a  familiar  variety  purchasable  in  every  seed  store.  It  occurs  in 
two  varieties,  the  zc'/n'/e  and  the  red.  Ricin,  a  protein  constituent  of  the  castor  bean,  also 
possesses  pronounced  agglutinating  properties.  Because  of  its  poisonous  nature  it  is,  how- 
ever, not  suitable  for  use  in  class  experiments. 

-Mendel:  Archivio  di  fisiologia,  7,  168,  1909;  Schneider:  Journal  Biol.  Chem.,  11,  47, 
1912. 

'  Rabbit's  blood  is  especially  desirable  (Mendel:  loc.  cil.)  and  may  be  obtained  for  the 
purpose  by  bleeding  from  a  small  cut  on  the  animal's  ear  and  detibrinating. 

*  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin. 

*  Alkaline  solutions  should  be  made  slightly  acid  with  acetic  acid,  as  the  blue  end- 
reaction  is  very  sensitive  to  alkali. 


BLOOD   AND    LYMPH  263 

these  conditions,  a  few  cubic  centimeters  of  alcohol  shovild  be  added  and  the  tube 
gently  shaken,  whereupon  a  blue  coloration  will  appear  in  the  upper  alcohol- 
turpentine  layer. 

A  control  test  should  always  be  made,  using  water  in  place  of  the  solution  under 
examination.  In  the  detection  of  very  minute  traces  of  blood  only  3-5  drops  of 
the  guaiac  solution  should  be  employed. 

13.  Ortho-tolidin  Test  (Ruttan  and  Hardisty).' — To  i  c.c.  of  a  4  per  cent 
glacial  acetic  acid  solution  of  o-tolidin-  in  a  test-tube  add  i  c.c.  of  the  solution 
under  examination  and  i  c.c.  of  3  per  cent  hydrogen  peroxide.  In  the  presence 
of  blood  a  bluish  color  develops  (sometimes  rather  slowly)  and  persists  for  some 
time  (several  hours  in  some  instances). 

This  test  is  said  to  be  as  sensitive  for  the  detection  of  occult  blood 
in  feces  and  stomach  contents  as  is  the  benzidine  reaction.  It  is  also 
claimed  to  be  more  satisfactory  for  urine  than  any  other  blood  test. 
The  acetic  acid  solution  may  be  kept  for  one  month  with  no  reduction 
in  delicacy. 

14.  Benzidine  Reaction.— This  is  one  of  the  most  delicate  of  the 
reactions  for  the  detection  of  blood.  Dififerent  benzidine  prepara- 
tions vary  greatly  in  their  sensitiveness,  however.  Inasmuch  as  ben- 
zidine solutions  change  readily  upon  contact  with  light  it  is  essential 
that  they  be  kept  in  a  dark  place. 

The  test  is  performed  as  follows :  To  a  saturated  solution  of  benzidine  in 
alcohol  or  glacial  acetic  acid  add  an  equal  volxmie  of  3  per  cent  hydrogen  peroxide 
and  I  c.c.  of  the  solution  vmder  examination.  If  the  mixture  is  not  already  acid 
render  it  so  with  acetic  acid,  and  note  the  appearance  of  a  green  or  blue  color. 
A  control  test  should  be  made  substituting  water  for  the  solution  under 
examination. 

The  hemoglobin  decomposes  the  hydrogen  peroxide  (catalysis)  and 
the  liberated  oxygen  oxidizes  the  benzidine.  The  sensitiveness  of  the 
benzidine  reaction  is  greater  when  applied  to  aqueous  solutions  than 
when  applied  to  the  urine.  According  to  Ascarelli^  the  benzidine  reac- 
tion serves  to  detect  blood  when  present  in  a  dilution  of  i  :  3,000,000. 
Walter^  has  also  shown  the  test  to  be  very  delicate,  and  claims  it  to  be 
more  satisfactory  than  the  guaiac  test. 

Lyle,    Curtman   and   Marshall  have  investigated   the   benzidine 

^  Rattan  and  Hardisty:  Canadian  Medical  Ass'n  Journal,  Nov.,  1912;  also  Biochemical 
Bull.,  2,  225,  1913. 

2  NHo  NHo 

\  ,     /       ■ 

CoH*  —  C0H4. 

/  \ 

CH3  CH3 

^  Ascarelli:  II  policlin  sez.  prat.,  1909. 
■•  Walter:  Detit.  vied.  Woclt.,  36,  p.  309. 
*Lyle,  Curtman  and  Marshall:  Jour.  Biol.  C//f»;.,   iq.  445,   19x4. 


264  PHYSIOLOGICAL   CHEMISTRY 

reaction  very  carefully.     They  suggest  a  new  procedure  in  preparing  the 
reagent^  and  in  conducting  the  test. 

The  test  follows:  Into  a  perfectly  clean  dry  test-tube  introduce  1.4  c.c. 
benzidine  solution, ^  add  0.2  c.c.  of  water  or  glacial  acetic  acid,  then  i  c.c.  of  the 
fluid  to  be  tested  and  finally  0.4  c.c.  of  3  per  cent  hydrogen  peroxide.  Note  the 
appearance  of  a  blue  color,  which  reaches  its  maximum  in  five  to  six  minutes. 

The  acetic  acid  keeps  the  benzidine  in  solution.  An  excess  dimin- 
ishes the  delicacy  of  the  reagent. 

Hydrogen  peroxide  supplies  oxygen  for  the  reaction  and  also  bleaches 
the  blue  color.  An  excess  of  peroxide  interferes  with  the  reaction  by 
destroying  the  catalytic  power  of  the  blood  and  by  reacting  with  the 
benzidine  itself,  with  the  formation  of  products  which  appear  to  have 
an  inhibitory  action.  It  is  very  essential  that  the  peroxide  be  added 
last. 

The  benzidine  solution  should  be  dilute.  Such  solutions  are  exceed- 
ingly sensitive  and  permit  the  detection  of  blood  when  present  in  ratio 
I  :  5,000,000. 

15.  Hem  in  Test. — (a)  Teichmann's  Method. — ^Place  a  very  small  drop  of 
blood  on  a  microscopic  shde,  add  a  minute  grain  of  sodiixm  chloride^  and  care- 
fully evaporate  to  dryness  over  a  low  flame.  Put  a  cover-glass  in  place,  nm 
imdemeath  it  a  drop  of  glacial  acetic  acid  and  warm  gently  until  the  formation  of 
gas  bubbles  is  noted.  Add  another  drop  of  glacial  acetic  acid,  cool  the  prepara- 
tion, examine  under  the  microscope  and  compare  the  crystals  with  those  shown  in 
Figs.  78  and  79. 

The  hemin  crystals  result  from  the  decomposition  of  the  hemoglobin 
of  the  blood.  What  are  the  steps  involved  in  this  process?  The  hemin 
crystals  are  also  called  Teichmann's  crystals.  Is  this  an  absolute  test 
for  blood?  Is  it  possible  to  differentiate  between  human  blood  and  the 
blood  of  other  species  by  means  of  the  hemin  test? 

(b)  Nippe's  Method. 3 — Spread  a  small  drop  of  blood  on  a  sUde  in  the  form  of 
a  film  and  evaporate  to  dryness  over  a  low  flame.  Now  add  2  drops  of  a  solution 
containing  o.i  gram  each  of  potassixim  chloride,  iodide  and  bromide  in  100  c.c. 
of  glacial  acetic  acid.  Place  a  cover-glass  in  position  and  heat  gently  over  a  low 
flame  until  gas  bubbles  form  and  the  solution  boils.  Run  1-2  drops  of  the 
reagent  underneath  the  cover-glass  and  examine  under  a  microscope.  Compare 
the  crystals  with  those  shown  in  Figs.  78  and  79. 

This  method  is  more  rapid  than  Teichmann's  method  and  crystals 

1  Benzidine  solution  may  be  prepared  as  follows:  Place  4.33  c.c.  of  glacial  acetic  acid  in 
a  small  Erlenmeyer  flask,  warm  to  50°  and  add  0.5  gram  of  benzidine.  Heat  the  flask  for 
eight  to  ten  minutes  in  water  at  50°.  To  the  resultant  solution  add  19  c.c.  of  distilled 
water.     This  solution  may  be  kept  for  several  days  without  deterioration. 

2  Buckmaster  considers  the  use  of  potassium  chloride  preferable. 
^Nippe:  Dent.  nied.  Woch.,  38,  2222,  1912. 


BLOOD    AND    LYMPH  265 


-^^  "^  ^  ^ 


^e:^"**^ 


Fig.  78. — Heshn  Crystals  from  Human  Blood. 

Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the  University 

of  Pennsylvania. 


Fig.  79. — Hemin  Crystals  from  Sheep  Blood. 

Reproduced  from  a  micro-photograph  furnished  by  Prof.  E.  T.  Reichert,  of  the 
University  of  Pennsylvania. 


266  PHYSIOLOGICAL   CHEMISTRY 

of  inorganic  chlorides  are  not  formed.     In  Teichmann's  method  crystals 
of  sodium  chloride  often  obscure  the  hemin  crystals. 

(c)  Atkinson  aiid  KendalVs  Method. — Introduce  a  small  amount  of  the  solution 
under  examination  into  a  tube  closed  at  one  end,  add  sodium  chloride  and  glacial 
acetic  acid  as  in  Teichmann's  method, ^  fuse  or  tightly  plug  the  open  end  of  the  tube 
and  heat  for  fifteen  minutes  in  a  boiling  water-bath. ^  Remove  the  tube  and  permit 
it  to  cool  to  room  temperature  spontaneously.  When  the  tube  has  cooled,  break 
it  open,  transfer  the  contents  to  a  watch  glass  or  small  evaporating  dish  and  con- 
centrate on  a  water-bath  until  the  volume  of  the  fluid  in  the  watch  glass  or  dish 
has  been  reduced  to  a  few  drops.  Transfer  a  drop  of  this  fluid  to  a  slide,  cover 
with  a  cover  slip,  allow  the  slide  to  stand  for  a  few  minutes  and  examine  it  under  a 
microscope.  Compare  the  crystals  with  those  shown  in  Figs.  78  and  79,  page  265. 
In  case  crystals  of  sodium  chloride  (see  Fig.  80)  obstruct  the  view  of  the  hemin 
crystals,  dissolve  the  sodium  chloride  crj'stals  by  running  a  drop  of  water  under 
the  cover  slip. 

((f)  V.  Zeynek  and  Nencki's  Method. — To  10  c.c.  of  defibrinated  blood  add  acetone 
until  no  more  precipitate  forms.  Filter  off  the  precipitated  protein  and  extract  it 
with  10  c.c.  of  acetone  made  acid  with  2-3  drops  of  hydrochloric  acid.  Place  a 
drop  of  the  resulting  colored  extract  on  a  slide,  immediately  place  a  cover-glass  in 
position  and  examine  under  the  microscope.  Upon  the  evaporation  of  the  acetone, 
crj'stals  of  hemin  will  form.  Larger  crystals  may  be  obtained  by  evaporating  the 
acetone  extract  about  one-half,  transferring  it  to  a  stoppered  vessel  and  allowing  it 
to  remain  overnight. 

(e)  Schalfijew's  Method. — Place  20  c.c.  of  glacial  acetic  acid  in  a  small  beaker 
and  heat  to  8o°C.  Add  5  c.c.  of  strained  defibrinated  blood,  again  bring  the 
temperature  to  8o°C.,  remove  the  flame  and  allow  the  mixture  to  cool.  Examine 
the  crystals  under  the  microscope  and  compare  them  with  those  reproduced  in 
Figs.  78  and  79,  page  265. 

16.  Catalytic  Action. — To  about  10  drops  of  blood  in  a  test-tube  add  twice  the 
volume  of  hydrogen  peroxide,  without  shaking.  The  mixture  foams.  What  is 
the  cause  of  this  phenomenon? 

17.  CrystaUization  of  Oxyhemoglobin.  Reichert's  Method. — Add  to  5  c.c. 
of  the  blood  of  the  dog,  horse,  guinea-pig,  or  rat,  before  or  after  laking,  or  de- 
fibrinating,  from  i  to  5  per  cent  of  ammonium  oxalate  in  substance.  Place  a 
drop  of  this  oxalated  blood  on  a  sHde  and  examine  under  the  microscope.  The 
crystals  of  oxyhemoglobin  wiU  be  seen  to  form  at  once  near  the  margin  of  the 
drop,  and  in  a  few  minutes  the  entire  drop  may  be  a  soUd  mass  of  crystals. 
Compare  the  crystals  with  those  shown  in  Figs.  70  to  76,  pages  251  to  254. 

18.  Preparation  of  Hematin. — Place  100  c.c.  of  hcmolyzed  ilaked)  blood  in  a 
beaker  and  add  95  per  cent  alcohol  until  precipitation  ceases.  What  bodies  are 
precipitated?  Transfer  the  precipitate  to  a  flask  and  boil  with  95  per  cent  alcohol 
previously  acidulated  with  sulphuric  acid.  Through  the  action  of  the  acid  the 
hemoglobin  is  spUt  into  hematin  and  a  protein  body  called  globin.  Later  the 
"sulphuric  acid  ester  of  hematin"  is  formed,  which  is  soluble  in  the  alcohol.  Con- 
tinue heating  until  the  precipitate  is  no  longer  colored,  then  filter.  Partly  saturate 
the  filtrate  with  sodium  chloride  and  warm.     In  this  process  the  "hydrochloric  acid 

1  Care  should  be  taken  not  to  add  too  great  an  excess  of  these  reagents. 
-  This  process  insures  constancy  of  temperature  and  strength  of  reagents. 


BLOOD  AND  LYMPH 


267 


ester  of  hematin"  is  formed.  Filter  and  dissolve  on  the  filter  paper  by  sodivim 
carbonate.  Save  this  alkaline  solution  of  hematin  and  make  a  spectroscopic  ex- 
amination later  after  becoming  familiar  with  the  use  of  the  spectroscope.  How 
does  the  spectrum  of  oxyhemoglobin  differ  from  that  of  the  derived  alkali  hematin  ? 
19.  Preparation  of  Thrombin  fHowell).' — Prepare  fibrin  from  pig's  blood 
according  to  directions  given  on  page  268.  Wash  the  fibrin  thoroughly  in  water 
to  remove  hemoglobin.  Squeeze  out  the  water,  mince  the  fibrin  and  cover  with 
an  8  per  cent  sodium  chloride  solution  and  allow  to  stand  in  the  cold  for  48  hours. 
Filter.  Precipitate  the  thrombin  fand  other  proteins  j  from  the  filtrate  by  adding 
an  equal  volume  of  acetone.  Filter  the  mixture  rapidly  through  a  nximber  of 
small  (25-50  c.c.)  filters.  Spread  out  filter  papers  and  precipitate  and  dry  rapidly 
in  a  current  of  cold  air.  Cut  the  dried  papers  into  small  pieces  and  treat  with  a 
volume  of  water  eqmvalent  to  66  per  cent  of  the  8  per  cent  NaCl  previously  used. 
Allow  to  stand  one -half  hour  and  filter.  Shake  the  filtrate  with  chloroform 
(10-15  c.c.  per  100  c.c.  filtrate)  until  on  settling  no  opalescence'is  developed  by 


Fig.  80. — SiiDir-M  Chloride. 


heating  a  portion  of  the  supernatant  fluid.  Decant  the  Uquid  and  evaporate 
on  watch  glasses  (2  c.c.  to  a  watch  glass)  in  a  current  of  air.  Thrombin  so  pre- 
pared may  be  kept  indefinitely  in  a  desiccator. 

20.  Variation  in  Size  of  Erythrocytes. — Prepare  two  small  funnels  with  filter 
papers  such  as  are  used  in  quantitative  analysis.  Moisten  each  paper  with  physio- 
logical (isotonic)  salt  solution.  Into  one  funnel  introduce  a  small  amount  of 
defibrinated  ox  blood  and  into  the  other  funnel  allow  blood  to  drop  directly  from  a 
decapitated  frog.  Note  that  the  filtrate  from  the  ox  blood  is  colored,  whereas  that 
from  the  frog  blood  is  colorless.  What  deduction  do  you  make  regarding  the 
relative  size  of  the  erythrocytes  in  ox  and  frog  blood?  Does  either  filtrate  clot? 
Why? 

n.  Blood  Serum- 

I.  Coagulation  Temperature. — Place  5  c.c.  of  undiluted  serum  in  a  test-tube 
and  determine  its  temperature  of  coagulation  according  to  the  method  described 

1  Howell:  Am.  Jour.   Physiol.,  32,  264.  igi3. 

*  For  directions  as  to  preparation  of  serum,  see  "Reagents  and  Solutions."     (Page  593.) 


268  PHYSIOLOGICAL    CHEMISTRY 

on  page  105.     Note  the  temperature  at  which  a  cloudiness  occurs  as  well  as  the 
temperature  at  which  coagulation  is  complete. 

2.  Precipitation  by  Alcohol. — To  5  c.c.  of  senun  in  a  test-tube  add  twice  the 
amount  of  95  per  cent  alcohol  and  thoroughly  mix  by  shaking.  What  is  this  pre- 
cipitate? Make  a  confirmatory  test.  Test  the  alcohoUc  filtrate  for  protein. 
Explain  the  result. 

3.  Proteins  of  Blood  Serum. — ^Place  about  10  c.c.  of  serum  in  a  small  evapo- 
rating dish,  dilute  with  5  c.c.  of  water  and  heat  to  boihng.  At  the  boiUng-point 
acidify  sUghtly  with  dilute  acetic  acid.  Of  what  does  this  coagulum  consist? 
Filter  off  the  coagulum  (reserve  the  filtrate)  and  test  it  as  follows : 

(a)  Millon's  Reaction. — Make  the  test  according  to  directions  given  on  page 

97. 

(b)  Hopkins-Cole  Reaction. — Make  the  test  according  to  directions  given  on 
page  98. 

4.  Sugar  in  Serum. — To  5  c.c.  of  the  neutralized  filtrate  from  Experiment  3 
add  5  drops  of  Fehling's  solution  and  boil  one  minute.    What  do  you  conclude? 

5.  Detection  of  Sodium  Chloride. — (a)  Test  a  little  of  the  filtrate  from  Ex- 
periment 3  for  chlorides,  by  the  use  of  nitric  acid  and  silver  nitrate,  (b)  Evapo- 
orate  5  c.c.  of  the  filtrate  from  Experiment  3  in  a  watch  glass  on  a  water-bath. 
Examine  the  crystals  and  compare  them  with  those  reproduced  in  Fig.  80,  p.  267. 

6.  Separation  of  Serum  Globulin  and  Senun  Albvunin. — Place  10  c.c.  of 
blood  serum  in  a  small  beaker  and  saturate  with  magnesimn  sulphate.  What  is 
this  precipitate?  Filter  it  off  and  acidify  the  filtrate  sUghtly  with  acetic  acid. 
What  is  this  second  precipitate?  Filter  this  precipitate  off  and  test  the  filtrate 
by  the  biuret  test.     What  do  you  conclude? 

III.  Blood  Plasma 

1.  Preparation  of  Oxalated  Plasma. — Allow  arterial  blood  to  run  into  an  equal 
volume  of  0.2  per  cent  ammonium  oxalate  solution. 

2.  Preparation  of  Fibrinogen. — To  25  c.c.  of  oxalated  plasma  add  an  equal 
volume  of  saturated  sodiiun  chloride  solution.  Note  the  precipitation  of  fibrin- 
ogen. Filter  off  the  precipitate  (reserve  the  filtrate)  and  test  it  by  a  protein  color 
test  (see  page  97). 

3.  Effect  of  Calcium  Salts. — Place  a  small  amount  of  oxalated  plasma  in  a 
test-tube  and  add  a  few  drops  of  a  2  per  cent  calciiim  chloride  solution.  What 
occurs?    Explain    it. 

4.  Preparation  of  Salted  Plasma. — Allow  arterial  blood  to  run  into  an  equal 
volume  of  a  saturated  solution  of  sodium  sulphate  or  a  10  per  cent  solution  of 
sodium  chloride.    Keep  the  mixture  in  a  cool  place  for  about  24  hours. 

5.  Effect  of  Dilution. — Place  a  few  drops  of  salted  plasma  in  a  test-tube  and 
dilute  it  with  10-15  volumes  of  water.     What  do  you  observe?    Explain  it. 

rV.  Fibrin 

I.  Preparation  of  Fibrin. — Allow  blood  to  flow  directly  from  the  animal  into 
a  vessel  and  rapidly  whip  it  by  means  of  a  bundle  of  twigs,  a  mass  of  strong  cords, 
or  a  specially  constructed  beater.  If  a  pure  fibrin  is  desired  it  is  not  best  to 
attempt  to  manipulate  a  large  volmne  of  blood  at  one  time.  After  the  fibrin  has 
been  collected  it  shoxild  be  freed  from  any  adhering  blood  clots  and  washed  in 
water  to  remove  further  traces  of  blood.  The  piure  product  should  be  very  Ught 
in  color.     It  may  be  preserved  under  glycerol,  dilute  alcohol,  or  chloroform  water. 


BLOOD    AND    LYMPH  269 

2.  Solubility.— ^Try  the  solubility  of  small  shreds  of  freshly  prepared  fibrin 
in  water,  dilute  acid  and  alkali. 

3.  Millon's  Reaction. — Make  the  test  according  to  directions  given  on  page 

97- 

4.  GlyoxyUc  Acid  Reaction  CHopkins-Colej. — Make  the  test  according  to 
directions  given  on  page  98. 

5.  Biuret  Test. — Make  the  test  according  to  directions  given  on  page  98. 

V.  Detection  of  Blood  in  Stains  on  Cloth,  Etc. 

1.  Identification  of  Corpuscles. — ^If  the  stain  under  examination  is  on  cloth 
a  portion  should  be  extracted  with  a  few  drops  of  glycerol  or  physiological  ^0.9  per 
cent)  soditim  chloride  solution.  A  drop  of  this  solution  should  then  be  examined 
under  the  microscope  to  determine  if  corpuscles  are  present. 

2.  Tests  on  Aqueous  Extract. — A  second  portion  of  the  stain  should  be 
extracted  with  a  small  amount  of  water  and  the  following  tests  made  upon  the 
aqueous  extract : 

(a)  Hemochromogen. — Make  a  small  amount  of  the  extract  alkaline  by 
potassiimi  hydroxide  or  sodiimi  hydroxide,  and  heat  imtil  a  brownish-green  color 
results.  Cool  and  add  a  few  drops  of  ammoniimi  sulphide  or  Stokes'  reagent 
(see  page  296)  and  make  a  spectroscopic  examination.  Compare  the  spectnmi 
with  that  of  hemochromogen  (see  Absorption  Spectra,  Plate  11).  Hankin^  has 
suggested  a  test  based  upon  the  formation  of  cyanhemochromogen  and  the 
microspectroscopical  demonstration  of  the  spectrum  of  this  compoimd. 

(b)  Hemin  Test. — Make  this  test  upon  a  small  drop  of  the  aqueous  extract 
according  to  the  directions  given  on  page  264. 

(c)  Guaiac  Test. — Make  this  test  on  the  aqueous  extract  according  to  the 
directions  given  on  page  262.  The  guaiac  solution  may  also  be  appUed  directly 
to  the  staia  without  previous  extraction  in  the  following  manner :  Moisten  the 
stain  with  water,  and  after  allowing  it  to  stand  several  minutes,  add  an  alcohoUc 
solution  of  guaiac  (strength  about  i  :  60)  and  a  httle  hydrogen  peroxide  or  old 
tvirpentine.     The  customary  blue  color  will  be  observed  in  the  presence  of  blood. 

(d)  Benzidine  Reaction. — Make  this  test  according  to  directions  given  on 
page  263. 

(e)  Acid  Hematin. — If  the  stain  fails  to  dissolve  in  water  extract  with  acid 
alcohol  and  examine  the  spectrum  for  absorption  bands  of  acid  hematin  (see 
Absorption  Spectra,  Plate  II). 

'  Hankin:     Brit.  Med.  Jour.,  p.  1261,  1906. 
Sutherland  and  Mitra:  Biochemical  Journal,  8,  128,  191 4. 


CHAPTER  XVI 

BLOOD  ANALYSIS 

The  study  of  the  composition  of  the  blood  under  various  normal  and 
pathological  conditions  has  received  great  impetus  from  the  development 
of  methods  for  blood  analysis  which  require  but  small  amounts  of 
material  and  yet  give  accurate  results.  Many  facts  of  physiological 
as  well  as  clinical  importance  have  thus  been  made  available.  Some 
t)^ical  examples  of  data  obtained  in  this  way  are  given  in  the  following 
table: 

COMPOSITION  OF  NORMAL  BLOOD  AND  OF  THE  BLOOD  IN  CERTAIN 
PATHOLOGICAL   CONDITIONS^ 


Normal 

Chronic 
nephritis 

Uremia 

Early 
diabetes 

Severe 
diabetes 

Gout 

Lipemia 

Cholelith- 
iasis 

Ar- 
thritis 

Total  solids. 

13-19 

12-18 

17-20 

19-21 

pel    CCUt *■-<■  .  w 

Total  N,  per  t 
cent 30 

2.5-3-0 

1.7-2.7 

I . 8-2 . 9 

Non-protein     1 
N i.    25-35 

35-90 

90-350 

25—35 

Urea  N ;      12-23 

16-70           70^300 

Uric  acid 

1-3 

1-4 

4-27 

3-6 

3-8 

Creatinine.. . . 

1-2 

1-3 

4-33 

Creatine 5-9 

S-30 

Amino-acidN.         4-5 

6-16.0! 

Ammonia  N.      0.1-0.2 

0.1-0.2 

0.2-1.0 

Sugar,  percent 

Acetone  + 
Acetoacetic 

0.06-0. II 

0.1-0.2 

0.15-0.30 

0.3-0.8 

2-25 

I. 5-12 

10-40 

^-hydroxy- 
butyric  acid   1       0-3 . 0 

5-25 

5-15 

lO-SO 

Cholesterol, 

0.17-0. 35 

o.is-0.30 

o.S-3.6 

0.28-0.95 

Chlorides      as 
NaCl,  percent      0.65 

0. 55-0. 75 

0 . 4S-0 . 65 

0.60 

• 

Acid      soluble] 
phosphorus..:       2-6 

3-7 

7-21 

Lipoid      phos- 
phorus          6-12 

8-13 

8-30 

! 

3-18 

3-29 

'  Results  are  expressed  as  milligrams  per  100  grams  of  blood  unless  otherwise  indicated. 
Some  of  the  figures  given  are  based  upon  but  few  analyses  and  may  not  be  entirely  charac- 
teristic. The  data  here  tabulated  have  been  compiled  from  the  work  of  many  observers. 
The  following  may  be  particularly  mentioned:  Myers  and  Fine:  Jour.  Biol.  Chem.,  20, 
391,  xgi 5;  Post-Graduate,  1914-15;  reprinted  as  "Chemical  Composition  of  the  Blood  in 
Health  and  Disease,"  New  York,  1915;  Folin  and  Denis:  Jour.  Biol.  Chem.,  14,  29,  1913; 
13,  469,  1913;  17,  487,  1914;  Arch.  Int.  Med.,  16,  33,  1915;  Christian,  Frothingham  and 
Wood:  Am.  J.  Med.  Sci.,  150,  655,  1915;  Greenwald:  Jour.  Biol.  Chem.,  21,  29,  1915;  Van 
Slyke  and  Meyer:  Jour.  Biol.  Chem.,  12,  399,  1912;  Bloor:  Jour.  Biol.  Chem.,  23,  317,  1915; 
Marriott:  Jour.  Biol.  Chem.,  16,  293,  1913;  18,  507,  1914. 

2  A  short  time  after  a  meal  rich  in  fat  the  blood  may  contain  considerably  more  fat. 

270 


BLOOD    ANALYSIS  27  I 

It  will  be  noted  that  in  chronic  nephritis  the  principal  change  is  in 
the  urea  and  non-protein  nitrogen  of  the  blood  which  may  increase 
considerably.  In  severe  cases  associated  with  uremia  the  retention  of 
these  forms  of  nitrogen  may  be  very  great  and  there  is  a  consequent 
rise  in  the  blood  content  which  may  amount  to  looo  per  cent  or  more. 
In  uremia  there  is  likewise  a  great  increase  in  other  individual  nitroge- 
neous  components  of  the  blood  such  as  uric  acid,  creatinine,  creatine, 
amino-acid  nitrogen,  and  even  of  ammonia.  The  increase  in  creatinine 
has  been  shown  by  Myers  and  Fine  and  others  to  be  significant,  inas- 
much as  this  increase  does  not  appear  to  occur  in  other  types  of  nephritis. 
Uric  acid  is  greatly  increased  in  uremia  and  may  be  very  much  higher 
than  in  gout.  Associated  with  uremia  there  is  ordinarily  an  acidosis. 
There  may  be  an  increase  in  the  sugar  of  the  blood  and  a  very  great 
increase  of  the  acetone  bodies  present.  An  increase  is  also  generally 
found  in  cholesterol  and  in  the  various  forms  of  phosphorus  of  the  blood. 

In  diabetes  the  most  noteworthy  changes  are  in  the  content  of  glucose 
and  of  acetone  bodies.  Glucose  may  be  increased  above  the  normal 
(about  0.1  per  cent)  to  0.15-0.80  per  cent.  The  increase  in  acetone, 
diacetic  acid  and  hydroxybutyric  acid  is  very  marked  in  comparison 
with  the  minute  amounts  found  in  normal  blood.  There  may  also  be 
an  increase  in  fat  and  other  lipoids  in  severe  diabetes. 

In  gout  the  characteristic  change  is  in  the  uric  acid  content  which  is 
almost  always  considerably  increased.  Other  forms  of  nitrogen  are 
affected  but  little.  In  arthritis  the  blood  may  also  be  high  in  uric  acid 
but  in  this  case  ordinarily  there  is  a  rise  in  non-protein  nitrogen  also. 

Lipemia  is  usually  associated  with  an  increased  sugar  content  of  the 
blood.  The  fat  content  in  this  condition  has  been  found  as  high  as  29 
per  cent.  There  is  a  correspondingly  large  increase  in  the  cholesterol 
of  the  blood. 

In  cholelithiasis  there  appears  generally  to  be  a  fairly  marked 
increase  in  the  cholesterol  content  of  the  blood  and  this  determination 
is  thus  of  diagnostic  aid.  Some  increase  may  also  be  found  in  other 
disorders  as  in  nephritis,  severe  diabetes,  pregnancy,  alteriosclerosis 
and  syphilis. 

Methods 

Non -protein  Nitrogen.—  (a)  Colorimetric  Method  of  Folin  and  Denis .^ 
— Principle. — This  method,  which  is  simple  and  convenient,  depends 
upon  the  removal  of  the  proteins  from  a  sample  of  blood  by  precipita- 
tion with  methyl  alcohol,  and  the  estimation  of  nitrogen  in  the  methyl 

*  Folin  and  Denis:  /.  Biol.  Client.,  11,  527,  191 2. 


272  PHYSIOLOGICAL   CHEMISTRY 

alcohol  solution  (after  the  removal  of  the  proteins)  by  means  of  oxi- 
dation and  Nesslerization.  The  details  of  the  procedure  are  carried 
out  in  the  following  manner: 

Method  of  Drawing  Blood. — ^Attach,  by  means  of  a  short  piece  of  pure  gum 
tubing,  an  hypodermic  needle  about  i  mm.  in  diameter  and  25  mm.  in  length 
(previously  sterilized  and  paraflined)  to  the  tip  of  a  2  or  5  c.c.  pipette.  Introduce 
into  the  upper  end  of  the  pipette  (which  must  be  perfectly  clean  and  dry)  a  small 
pinch  of  powdered  potassium  oxalate,  and  allow  it  to  run  down  into  the  tip  and  the 
needle.  Attach  a  piece  of  rubber  tubing  to  the  upper  end  of  the  pipette,  and  to  this 
a  mouthpiece  consisting  of  a  short  tapering  glass  tube.  Place  a  pinchcock  over 
the  rubber  tube  near  the  top  of  the  pipette.  To  draw  the  blood,  insert  the  needle 
into  the  vein  or  artery  and  regulate  the  flow  by  means  of  the  pinchcock  and  suc- 
tion. The  exact  quantity  of  blood  desired  is  thus  obtained  without  any  waste  or 
clotting. 

Method  of  Isolating  Non-protein  Nitrogen  Constituents. — Methyl  alcohol  and 
zinc  chloride  are  employed  as  precipitants  for  the  protein  materials  of  the  blood, 
and  the  determination  of  the  non-protein  nitrogen  is  then  carried  out  upon  a 
portion  of  the  methyl  alcohol  extract.  The  procedure  is  as  follows:  Transfer 
the  blood,  as  soon  as  drawn,  to  a  measuring  flask  which  is  half  filled  with  pure 
methyl  alcohol  (must  be  acetone  free).  Fill  to  the  mark  with  methyl  alcohol 
and  shake  thoroughly.  (If  2  c.c.  of  blood  are  taken,  25  c.c.  flasks  are  used  for  the 
precipitation,  while  for  5  c.c.  of  blood  50  c.c.  flasks  are  used.)  Allow  the  flask 
to  stand  for  at  least  two  hours  and  at  the  end  of  that  time,  or  later,  filter  the  con- 
tents through  dry  filter  paper.  Add  2-3  drops  of  a  saturated  alcoholic  solution  of 
zinc  chloride  to  the  filtrate  and  filter  again  through  a  dry  filter  paper  after  a  few 
minutes.  The  zinc  chloride  brings  down  an  appreciable  precipitate  and  the  last 
traces  of  coloring  matter,  so  that  the  second  filtrate  obtained  is  perfectly  colorless 
and  clear.    This  filtrate  is  used  for  the  determination  of  non-protein  nitrogen. 

Trichloracetic  Acid  Modification. — Greenwald^  has  suggested  the 
use  of  trichloracetic  acid  as  the  precipitant  for  the  proteins  of  the  blood, 
as  being  more  satisfactory  than  the  methyl  alcohol  and  zinc  chloride. 
The  objection  to  the  methyl  alcohol  is  that  some  of  the  amino-acids 
(creatine,  asparagine,  and  tyrosine)  are  insoluble  in  it  and  hence  pre- 
cipitated along  with  the  proteins.  These  acids  are  not  removed  by  the 
trichloracetic  acid.  Certain  nitrogenous  lipoid  substances  are  precipi- 
tated by  the  trichloracetic  acid  and  not  by  the  methyl  alcohol.  Green- 
wald  suggests  that  these  substances,  even  though  non-protein  in  char- 
acter, should  not  be  included  with  the  non-protein  nitrogen  of  the 
amino-acids  and  urea. 

Procedure. — ^Dilute  the  blood  to  ten  times  its  original  volume  with  2.5  per 
cent  trichloracetic  acid  solution.  Let  stand  30  minutes  and  then  filter.  Shake 
the  filtrate  with  about  4  grams  of  kaolin  per  100  c.c.  and  filter  again.  An  aliquot 
of  this  final  filtrate  is  taken,  digested  with  sulphuric  acid  and  nitrogen  deter- 
mined in  the  usual  way. 

^  Greenwald:  /.  Biol.  Chem.,  21,  61,  1915. 


BLOOD    AXALYSIS  273 

Determination  of  Total  Non -protein  Nitrogen. — Transfer  5  c.c.  of  the  alcoholic 
filtrate  to  a  large  Jena  test-tube  of  the  same  kind  as  is  used  in  urine  analysis 
(see  page  485).  Add  i  drop  of  concentrated  sulphuric  acid,  i  drop  of  kerosene, 
and  a  small  pebble  or  glass  bead  to  prevent  bimiping.  Immerse  the  test-tube 
in  a  beaker  of  boiUng  water  for  five  or  ten  minutes  to  drive  off  the  methyl  alcohol. 
When  the  alcohol  is  removed  add  i  c.c.  of  concentrated  sulphuric  acid,  i  gram  of 
potassium  sulphate  and  i  drop  of  copper  sulphate  solution.  Boil,  cool,  dilute  and 
aerate  the  solution  as  described  in  the  determination  of  total  nitrogen  in  urine 
(see  page  485),  except  that  the  ammonia  is  collected  in  a  large  test-tube  instead 
of  the  100  c.c.  flask.  Nesslerize  the  solution,  using  7  to  8  c.c.  of  diluted  Nessler 
reagent  (dilution  i  :5),  dilute  to  25  or  50  c.c.  according  to  the  amount  of  color, 
and  compare  with  a  standard  solution  containing  i  mg.  of  ammonia  nitrogen, 
Nesslerized  and  diluted  to  100  c.c.  and  the  colorimeter  prism  set  at  20  mm. 

Calculations. — If  5  c.c.  of  blood  are  diluted  to  50  c.c.  and  10  c.c.  of  the  alcohoUc 
extract  (equivalent  to  i  c.c.  of  blood)  are  used  for  the  determination,  the  amount 
of  non-protein  nitrogen  (as  milligrams  per  100  c.c.  of  the  blood)  can  be  obtained 

20 

by  use  of  the  formula  p  X  D,  in  which  R  stands  for  the  reading  of  the  unknown 

and  D  represents  the  volimie  to  which  its  ammonia  has  been  diluted.     If  the 
equivalent  of  0.4  c.c.  of  blood  has  been  taken  for  the  determination  the  formula 

P   X  D  is  used,  and  if  the  equivalent  of  0.5  c.c.  of  blood  has  been  taken  the 

40 
formula  becomes    ^  X  D. 

(b)  Colorimelric  Method  for  Finger  Blood. — Principle. — Taylor  and 
Hulton^  have  suggested  a  modification  for  the  determination  of  non- 
protein nitrogen  using  from  4  to  8  drops  of  blood.  It  is  based  upon  the 
observation  of  Gulick^  that  the  presence  of  small  amounts  of  potassium 
sulphate  does  not  appreciably  interfere  \\dth  the  Nesslerization  of  solu- 
tions containing  ammonium  salts.  The  Nesslerization  is  accordingly 
carried  out  directly  upon  the  oxidized  material  without  removal  of  the 
ammonia  by  aspiration  or  distillation.  The  authors  use  a  mixture  of 
ether  and  alcohol  for  the  precipitation  of  proteins.  The  authors  claim 
only  approximate  accuracy  for  the  method  and  the  small  amount  of 
lipoid  nitrogen  included  with  the  other  non-protein  nitrogen  may  be 
disregarded. 

Procedure. — Place  10  c.c.  of  a  mixture  of  absolute  alcohol  and  ether  (3  :i) 

in  a  small  Erlenmeyer  flask.     Stopper  and  weigh  the  flask  with  its  contents. 

Remove  the  stopper  and  allow  from  4  to  8  drops  of  blood  (depending  upon  the 

amount  of  non-protein  nitrogen)  to  drop  from  the  finger  which  has  been  cleaned 

and  pricked  with  a  sharp  lance.     The  blood  should  drop  freely  when  the  hand  is 

held  down.     Insert  the  stopper  and  weigh  the  flask  and  contents.     The  increase 

in  weight  is  of  course  the  weight  of  blood.     Within  a  half-hour  filter  the  mixture 

into  the  digestion  flask  and  wash  the  filter  with  5  c.c.  of  alcohol-ether.     The 

filtrate  is  clear  and  practically  free  from  protein,  although  it  probably  contains 

traces  of  nitrogen-containing  Upoids.     The  Kjeldahl  digestion  is  carried  out  in  a 

^  A.  E.  Taylor  and  F.  Hulton:  /.  Biol.  Chan.,  22,  63,  1915. 
*Gulick:  /.  Biol.  Clicm.,  18,  541,  1914. 

iS 


2  74  PHYSIOLOGICAL   CHEMISTRY 

25  c.c.  long-necked,  round-bottomed  Kjeldahl  flask,  the  neck  of  which  has  a 
crook  near  the  top.  Add  a  small  piece  of  acid  potassiimi  sulphate,  several  glass 
beads,  and  drive  off  the  alcohol-ether  by  heating  on  a  hot  plate.  When  the  flask 
is  nearly  dry  add  i  c.c.  of  concentrated  sulphuric  acid  and  not  more  than  300  or 
400  mg.  of  potassium  sulphate.  Then  place  the  flask  over  the  direct  flame  of  a 
micro-burner  (resting  the  bottom  of  the  flask  upon  an  asbestos  board  which  has  a 
circular  perforation  a  Uttle  smaller  than  the  circle  of  i  c.c.  of  sulphuric  acid  in  the 
digestion  flask)  and  heat  until  the  mixture  is  colorless.  Transfer  the  digestion 
mixture  to  a  100  c.c.  flask,  neutraUze  the  sulphuric  acid  with  about  3  c.c.  of  a  30 
per  cent  solution  of  sodium  hydroxide,  fiU  the  flask  to  over  three-quarters  full 
with  ammonia-free  water,  and  add  5  c.c.  of  Winkler's  modification  of  Nessler's 
solution  (see  page  603).  Dilute  to  mark  with  water  and  compare  the  color  in  the 
Duboscq  colorimeter  (see  Fig.  153,  p.  486)  against  a  standard  solution  of  ammo- 
nium sulphate. 

2.  Urea. — The  Urease  Method. — Van  Slyke  and  Cullen's^  Modification  of 
Marshall's  Method.- 

Principle. — See  Urease  Method,  Chapter  XXVI. 

Procedure. — Run  3  c.c.  of  fresh  blood  (carefully  measured  with  an  accurate 
pipette)  into  a  100  c.c.  test-tube  containing  i  c.c.  of  a  3  per  cent  solution  of  potas- 
sium citrate  (to  prevent  clotting).  Add  0.5  c.c.  of  the  urease  solution^  and  2  or  3 
drops  of  capryUc  alcohol  (to  prevent  foaming).  After  ten  minutes  add  15  c.c. 
of  a  saturated  solution  of  potassium  carbonate,  and  drive  off  the  ammonia  by 
aspiration  into  another  tube  containing  15  c.c.  of  hundredth -normal  hydrochloric 
or  sulphuric  acid.  Titrate  the  excess  of  acid  with  hundredth-normal  sodium 
hydroxide  or  potassium  hydroxide,*  using  methyl  red  or  ahzarin  as  indicator. 

Calculations. — Each  cubic  centimeter  of  acid  neutralized  indicates  o.oi  gram 
of  urea  per  100  c.c.  of  blood,  or  0.0467  gram  of  urea  nitrogen  per  100  c.  c.  of  blood. 
In  case  the  blood  should  be  one  of  the  rare  samples  containing  over  o  .15  per  cent 
of  urea,  all  the  acid  will  be  neutraUzed,  and  it  will  be  necessary  to  repeat  the 
determinations,  using  in  the  determination  only  i  c.c.  of  blood.  Fresh  blood 
contains  so  little  ammonia  that  it  may  be  disregarded.  For  further  discussion  of 
the  urease  method  see  Chapter  XXVI. 

3.  Uric  Acid. — Colorimetric  Microchemical  Method. — Principle. — 
Folin  and  Denis^  were  the  first  to  apply  the  technic  of  their  method 
for  the  determination  of  small  quantities  of  uric  acid  to  the  quan- 
titative estimation  of  uric  acid  in  blood.  (For  a  discussion  of  the 
principle  of  the  color  reaction  see  the  determination  of  uric  acid  in  urine, 
page  510.)  The  procedure  necessary  for  the  determination  in  blood  is 
somewhat  different  from  that  used  for  the  determination  in  urine  because 
of  the  presence  of  proteins  in  the  blood.  These  must  be  removed  first, 
and  then  the  uric  acid  may  be  determined  in  the  protein-free  extract, 
according  to  the  procedure  used  for  the  determination  in  urine.     The 

^  Van  Slyke  and  CuUen:  J.  Am.  Med.  Ass'n,  62,  1558,  1914. 
-Marshall:  Jour.  Biol.  Chem.,  15,  487,  1913. 

^  The  enzyme  solution  is  prepared  as  described  under  "Reagents  and  Solutions,"  p.  594. 
*  Rose  and  Coleman  (Biochem.  Bull.,  3,  411,  1914)  suggest  the  colorimetric  determina- 
tion of  the  ammonia  (see  page  492). 

'  Folin  and  Denis:  /.  Biol.  Chem.,  13,  469,  1913. 


BLOOD   ANALYSIS  275 

proteins  are  removed  from  the  blood  by  precipitation  with  hot  dilute 
acetic  acid  in  the  following  manner: 

(a)  Folin-Denis  Procedure. — The  blood  is  drawn  into  small,  wide-mouthed 
bottles  previously  weighed  and  containing  a  small  amount  (about  o.i  gram  1  of 
finely  powdered  potassium  oxalate.  From  the  subsequent  weight  of  each  bottle 
is  obtained  the  weight  of  the  blood.  Five  times  this  weight  of  N  100  acetic  acid 
solution'  is  transferred  to  an  ordinary  1000  c.c.  fiask  and  heated  to  boiling.  The 
oxalated  blood  is  then  poured  into  this  boiUng  acetic  acid  solution,  stirring  con- 
stantly, and  the  heating  is  continued  until  the  solution  has  again  begun  to  boil. 
The  mixture  is  filtered  while  stiU  hot.  The  coagulated  material  on  the  filter  paper 
is  transferred  back  into  the  flask  (by  means  of  a  small  spoon  or  a  spatula  1,  about 
200  c.c.  of  boiUng  water-  are  poured  over  it  and  it  is  allowed  to  stand  for  five 
minutes.  This  mixture  is  then  filtered  through  the  same  filter  as  was  used  for  the 
first  filtration.  The  filtrate  in  the  receiving  flask  should  be  very  nearly  as  clear 
as  water,  and  will  be  found  to  be  so  if  the  original  blood  was  promptly  shaken 
with  the  oxalate,  so  that  no  clotting  has  taken  place. 

If  clotting  has  occurred,  the  coagulation  and  washing  of  the  blood  is  a  Uttle 
more  complicated.  The  clot  leads  to  so  much  bumping  in  the  boiling  acetic  acid 
solution  that  it  is  not  practical  or  safe  to  try  to  heat  the  mixture  to  boiUng. 
.The  filtration  is,  therefore,  made  earUer.  The  partially  coagulated  clot  is  then 
broken  up  with  a  glass  rod,  transferred  to  a  mortar  and  there  ground  into  a  paste 
in  the  presence  of  hot  water.  This  suspension  is  then  poured  on  the  filter.  The 
protein  material  on  the  filter  is  then  washed,  as  before,  with  about  200  c.c.  of  hot 
water.  In  this  case  the  combined  filtrates  are,  however,  never  colorless  but  more 
or  less  reddish.  On  being  heated  to  boiling  a  second  small  coagulum  will  be 
obtained  and  the  filtrate  will  then  be  practically  as  clear  as  water. 

The  combined  filtrate  and  washings,  containing  the  uric  acid  and  other  solu- 
ble materials,  is  further  acidified  by  the  addition  of  5  c.c.  of  50  per  cent  acetic 
acid  and  is  evaporated,  over  a  free  flame  in  a  suitable  dish,"  to  a  very  small 
volume  (about  3  c.c. ).  The  Uquid  is  then  poured  into  an  ordinary  centrifuge  tube 
and  the  dish  washed  with  two  successive  portions  of  o.i  per  cent  lithium  car- 
bonate solution,  using  about  2  c.c.  for  each  rinsing.  Any  solid  material  adhering 
to  the  sides  of  the  dish  is  removed  by  rubbing  with  a  rubber-tipped  stirring  rod. 
This  solid  material  can  be  removed  by  centrifuging  and  pouring  the  superna- 
tant Hquid  into  another  tube,  washing  the  sediment  with  lithium  carbonate  solu- 
tion (Smith). 

The  liquid  in  the  centrifuge  tube,  which  at  this  stage  should  not  be 
more  than  lo  c.c.  in  volume,  is  then  treated  as  in  the  method  for  urine. 

(b)  Benedict  Procedure. — Benedict^  has  suggested  that  the  pre- 
cipitation of  proteins  by  the  method  of  Folin  and  Denis  is  incomplete, 
and  proposes  the  use  of  colloidal  iron  for  the  completion  of  precipitation 

'  Prepared  by  diluting  0.6  c.c.  of  glacial  acetic  acid  to  i  liter. 

-  For  this  washing,  water  is  used  rather  than  .V/ioo  acetic  acid,  because  if  the  latter  is 
used  the  coagulum  will  give  off  more  or  less  of  the  blood  pigment  and  the  filtrates  are  less 
clear. 

'  Deep  (half  globular)  dishes  10  cm.  in  diameter  and  having  a  capacity  of  250  c.c.  are 
very  good  for  this  purpose.  While  free  flames  are  the  most  convenient  for  concentrating 
the  uric  acid  solutions,  care  must,  of  course,  be  taken  not  to  char  the  contents  toward  the 
end  of  the  operation.  Unless  the  solution  can  be  watched  carefully  at  this  stage,  it  is  safer 
to  linish  the  concentration  on  the  water-bath. 

*  Benedict:  /.  Biol.  Clicm.,  20,  629,  1915. 


276  PHYSIOLOGICAL   CHEMISTRY 

after  coagulation  in  dilute  acetic  acid.  Myers  and  Fine^  suggest  the 
use  of  aluminium  cream  for  this  purpose.  Benedict's  procedure  is  as 
follows: 

To  20  C.C.2  of  oxalated  blood  are  added  100  c.c.  of  boiling  N/ioo  acetic  acid 
in  a  casserole,  and  the  mixture  is  heated  to  boiling  for  a  moment.  Remove  the 
casserole  from  the  flame  and  add  200  c.c.  of  boiling  distilled  water.  Povir  the 
mixture  upon  a  folded  filter  and  wash  the  residue  with  50  c.c.  of  boiling  water 
(heated  in  the  same  casserole  in  which  the  original  coagulation  took  place). 
Transfer  the  whole  filtrate  to  a  casserole  and  boil  down  rapidly  to  a  volimie  of 
about  25  c.c.  Pour  this  solution  into  a  small  flask  roughly  marked  to  indicate  a 
volume  of  50  c.c.  Transfer  the  contents  of  the  casserole  to  the  flask  quantita- 
tively, with  the  help  of  two  or  three  portions  of  water,  heating  vigorously  to 
boiling  and  rubbing  the  sides  of  the  casserole  with  a  rubber-tipped  stirring  rod 
each  time.  The  total  volume  in  the  flask  should  not  exceed  50  c.c.  after  the  ad- 
dition of  the  washings.  Thoroughly  cool  the  tixrbid  solution  in  the  flask  imder 
running  water,  and  add  2  c.c.^  of  colloidal  iron  solution  (Merck's  "Dialyzed  Iron," 
5  per  cent  solution)  while  the  flask  is  being  gently  rotated.  Filter  the  mix- 
ture through  a  small  folded  filter  into  a  100  c.c.  Jena  Florence  flask,  and  wash 
the  residue  twice  with  distilled  water.  The  filtrate  obtained  here  should  be  as 
clear  and  colorless  as  distilled  water.  Boil  the  solution  down  to  a  volume  of  from 
I  to  2  c.c.  (care  being  taken  in  the  early  stages  to  prevent  bimiping),  then  care- 
fully pour  into  a  small  centrifuge  tube  and  wash  out  the  flask  with  three  portions 
of  water  (i  or  2  c.c.  each),  heating  each  to  boiling  in  the  flask  and  shaking  thor- 
oughly prior  to  transferring  it  to  the  centrifuge  tube.  The  volume  of  liqmd  in  the 
tube  at  this  point  should  be  from  5  to  10  c.c.  Cool  the  Uquid,  add  20  drops  of  the 
ammoniacal  sUver  magnesium  solution  and  proceed  with  the  determination  as 
described  under  Urine  (Chapter  XXVI).  If  the  amoimt  of  uric  acid  present  is 
very  small  the  addition  of  i  drop  of  cyanide  solution,  1  c.c.  of  uric  acid  re- 
agent, 5  c.c.  of  20  per  cent  sodivun  carbonate  solution,  and  dilution  to  25  c.c. 
are  carried  out  rather  than  using  the  larger  quantities  given  for  the  determina- 
tion in  the  urine. 

4.  Creatine  and  Creatinine.  Methods  oJFolin* — Preformed  Creatinine. — Meas- 
ure 10  c.c.  of  blood  into  a  50  c.c.  volumetric  flask  or,  better,  into  a  50  c.c.  shaking 
cylinder  which  can  be  closed  with  a  glass  stopper.  Fill  to  the  50  c.c.  mark  with  satu- 
rated picric  acid  solution  and  shake  a  few  times.  Add  about  i  gram  of  dry  picric 
acid  to  the  mixture  and  shake  for  five  minutes.  Transfer  the  mixture  to  centrifuge 
tubes,  throw  down  the  sediment  and  precipitate  and  pour  the  supernatant  liquid 
through  a  filter.  This  is  the  most  economical  process  where  but  little  blood  is  avail- 
able. If  desired,  however,  double  quantities  of  blood  and  reagents  may  be  taken  and 
filtration  carried  out  without  preliminary  centrifugation.  This  process  removes  the 
protein  materials  and  leaves  the  creatine  and  creatinine  in  the  filtrate  which  is  a  satu- 

1  Myers  and  Fine:  "Blood  in  Health  and  Disease,"  1915,  p.  14. 

^  Smaller  amounts  of  blood  may  be  employed,  and  the  quantity  of  acetic  acid  and 
water  correspondingly  reduced.  Unless  the  quantity  of  uric  acid  present  is  very  large, 
the  results  are  far  more  accurate  when  20  c.c.  of  blood  are  used. 

*  With  old  samples  of  blood  it  may  be  necessary  to  add  3  or  4  c.c.  of  the  iron  solution 
and  a  little  10  per  cent  sodium  chloride  solution.  When  the  precipitate  separates  in  large 
flocculent  masses  the  right  amount  of  iron  has  been  added.  Any  excess  of  iron  must  be 
avoided,  as  it  would  oxidize  some  of  the  uric  acid  later  on  in  the  process. 

*  Folin:  Jour.  Biol.  Chetn..  17,  475,  1914. 


BLOOD   ANALYSIS  277 

rated  picric  acid  solution.  The  preformed  creatinine  is  then  determined  colorimet- 
rically.  For  this  purpose  a  standard  solution  of  creatinine  for  comparison  is 
necessary.  Prepare  this  from  the  standard  creatinine  stock  solution  as  used  in  the 
analysis  of  urine  (see  chapter  on  Quantitative  Analysis  of  Urine)  by  diluting  an 
amount  of  this  solution  equivalent  to  i  mg.  of  creatinine  to  500  c.c.  with  saturated 
picric  acid  solution.  We  have  then  a  standard  solution  containing  0.2  mg.  of 
creatinine  in  100  c.c-  of  saturated  picric  acid  solution. 

Take  20  c.c.  portions  each  of  the  filtrate  and  of  the  standard  solution.  To  each 
solution  then  add  exactly  i  c.c.  of  10  per  cent  NaOH  from  a  burette.  (If  the  blood 
filtrate  becomes  turbid  on  addition  of  alkali  it  must  be  centrifuged  or  filtered.) 
Allow  to  stand  for  10  minutes  and  compare  the  colors  directly  in  the  colorimeter 
without  further  dilution.  The  standard  creatinine  solution  may  be  set  advantage- 
ously at  20  mm.,  although  this  is  not  necessary. 

•  Calculation. — Since  the  blood  was  diluted  five  times  in  the  precipitation  pro- 
cedure and  as  the  standard  for  comparison  contains  0.2  mg.  of  creatinine  per  100  c.c, 
it  is  merely  necessary  to  divide  the  reading  of  the  standard  by  the  reading  of  the 
unknown  to  obtain  without  further  calculation  the  number  of  milligram';  of  creatin- 
ine in  100  c.c.  of  blood. 

Creatine  Plus  Creatinine. — For  determining  the  total  creatinine  plus  creatine 
in  the  blood  carry  out  the  preliminary  precipitation  with  picric  acid  just  as  in  the 
determination  of  creatinine  above.  Take  10  c.c.  of  this  filtrate  for  the  determina- 
tion. Transfer  it  to  a  small  Erlenmeyer  flask  or  large  test-tube.  Cover  the  flask 
or  test-tube  with  tin  foil,  transfer  to  an  autoclave  and  heat  to  about  i2o°C.  for 
about  20  minutes.  The  autoclave  should  not  be  opened  until  the  temperature  has 
fallen  below  ioo°C.  Cool  the  solution  to  room  temperature,  rinse  into  a  25  c.c. 
volumetric  flask  with  saturated  picric  acid  solution.  Add  1.25  c.c.  of  10  per  cent 
NaOH  for  the  development  of  the  color. 

On  account  of  the  variations  in  the  creatine  content  of  normal  blood  two 
standard  creatinine  solutions  are  used.  In  working  on  pathological  cases  a  third 
standard  is  desirable.  These  standards  contain  0.5,  i,  and  2  mg.  of  creatinine 
respectively  per  100  c.c.  of  saturated  picric  acid  solution.  To  20  c.c.  of  each  of 
these  solutions  in  measuring  cylinders  add  i  c.c.  of  10  per  cent  NaOH  and  allow 
to  stand  for  10  minutes.  By  inspection  determine  which  standard  corresponds 
most  nearly  in  color  with  the  unknown  and  use  this  for  comparison.  The  standard 
is  usually  set  at  10  mm.  in  the  Duboscq  colorimeter. 

Calculation. — Multiply  the  reading  of  the  standard  by  125  and  by  0.5,  i,  or  2, 
according  to  which  standard  is  used,  and  divide  by  the  reading  of  the  unknown  in 
millimeters.  The  result  gives  the  number  of  milligrams  of  creatine  +  creatinine 
in  100  c.c.  of  the  blood  examined. 

5.  Amino-acid  Nitrogen,  (o)  Method  of  Van  Slyke  and  Meyer.^ — Principle. — 
The  protein  of  the  blood  is  removed  by  precipitation  with  alcohol  and  the  amino- 
acid  nitrogen  determined  in  the  filtrate  by  the  nitrous  acid  method. 

Procedure. — Thirty  to  50  c.c.  of  freshly  drawn  blood  are  mixed  with  9  or  10 
volumes  of  95  per  cent  alcohol  to  precipitate  the  proteins.  The  volume  of  the 
alcohol-blood  mixture  must  be  known,  but  in  case  it  is  not  convenient  to  use  a 
graduated  cylinder  for  the  mixture,  its  volume  can  be  taken  as  the  sum  of  the 
volumes  of  the  alcohol  and  blood  without  essentially  affecting  the  results.  The 
alcohol  and  blood  are  thoroughly  mixed,  the  vessel  containing  them  is  dosed  and 

'  Van  Slyke  and  Meyer:  Jour.  Biol.  Client.,  12,  399,  1912. 


278  PHYSIOLOGICAL   CHEMISTRY 

24  hours  are  allowed  for  precipitation  of  the  proteins  to  become  complete.  The 
solution  is  filtered  through  a  dry  folded  filter  into  a  measuring  cylinder  without 
washing  the  precipitate.  The  volume  of  filtrate  is  noted  and  is  taken  for  analysis 
as  an  aliquot  part  of  the  total  blood-alcohol  mixture.  The  filtrate  is  then  con- 
centrated to  a  volume  of  3-5  c.c.  and  used  for  determination  of  amino  nitrogen  by 
the  Van  Slyke  nitrous  acid  method  (see  Chapter  IV  on  Proteins).  The  use  of  a 
few  drops  of  caprylic  alcohol  to  prevent  foaming  is  advisable. 

(b)  Method  of  Cotistantino. ^^This  is  based  on  the  formol  titration  procedure. 
One  hundred  c.c.  of  blood  or  serum  is  mixed  with  a  measured  (500  c.c.)  volume  of  2 
per  cent  mercuric  chloride  solution  containing  0.8  per  cent  hydrochloric  acid.  The 
mixture  is  shaken  vigorously  in  a  stoppered  flask  and  allowed  to  stand  a  few  hours. 
Centrifugate  for  10  minutes,  pour  the  supernatant  liquid  through  a  dry  filter  into  a 
graduated  cylinder.  An  aliquot  of  the  filtrate  is  taken,  the  mercury  is  removed 
with  hydrogen  sulphide  and  the  latter  by  a  current  of  air.  The  liquid  is  exactly 
neutralized  and  concentrated  on  the  water-bath,  or  better,  at  50°  in  a  vacuum, 
MgO  added,  and  the  mixture  distilled  in  a  vacuum  at  45°  to  get  rid  of  ammonia. 
The  volume  should  now  be  about  30  c.c.  A  little  solid  barium  chloride  and  barium 
hydroxide  are  added  and  1.5  c.c.  of  0.5  per  cent  solution  of  phenolphthalein. 
Filter.  Neutralize  accurately  to  sensitive  litmus  paper.  Add  neutral  formalin 
solution  and  titrate  with  N/5  NaOH  as  described  in  the  chapter  on  quantitative 
analysis  of  urine  (page  504). 

6.  Ammonia. — Method  of  Folin  and  Denis. — The  determination  of  ammonia  in 
blood  is  attended  with  considerable  difficulty  because  of  the  fact  that  it  is  present 
only  in  very  small  amounts,  and  to  the  fact  that  blood  very  readily  and  quickly 
undergoes  changes  which  are  accompanied  by  the  formation  of  ammonia  from  the 
nitrogenous  compounds  present  in  the  fresh  blood. 

Of  the  methods  proposed  for  its  quantitative  estimation  that  of  Folin  and  Denis^ 
is  perhaps  least  unsatisfactory. 

Principle. — The  method  is  based  upon  the  liberation  of  the  ammonia  from  fresh 
blood  by  aspiration  after  adding  sodium  carbonate  solution,  and  the  Nesslerization 
of  the  solution  into  which  the  ammonia  is  aspirated. 

Procedure. — Place  10  c.c.  of  systemic  blood  or  5  c.c.  of  portal  or  mesenteric  blood^ 
in  a  large  Jena  test-tube.  Add  2  or  3  c.c.  of  a  solution  composed  of  15  per  cent 
potassium  oxalate  and  10  per  cent  sodium  carbonate,  and  about  5  c.c.  of  toluene. 
Connect  the  tube  for  aspiration  as  in  the  method  for  urea  in  blood;  start  the  air 
current,  and  run  as  fast  as  the  apparatus  wUl  stand  for  20  to  30  minutes.  Collect 
the  ammonia  in  another  test-tube  containing  i  c.c.  of  water  and  5  or  6  drops  of 
tenth-normal  acid. 

At  the  end  of  the  aspiration  Nesslerize  in  the  usual  manner  (see  method  for  non- 
protein nitrogen,  page  273)  but  more  cautiously,  adding  in  all  not  over  i  c.c.  of  the 
previously  diluted  Nessler  reagent  (dilution  1:5).  Transfer  the  contents  of  the 
receiver  to  a  10  c.c.  volumetric  flask,  fill  to  the  mark  with  ammonia-free  water,  and 
mix.  Fill  a  100  mm.  polariscope  tube  with  the  mixture  and  close  as  for  ordinary 
polariscopic  work. 

Prepare  two  standard  solutions,  one  containing  0.5  mg.  and  the  other  i.o  mg. 

of  nitrogen,  these  being  Nesslerized  simultaneously  with  the  unknown  solution  and 

made  up  to  volume  (100  c.c).     Use  the  standard  possessing  a  tint  most  similar  to 

that  of  the  unknown.     Compare  in  a  Duboscq  colorimeter,  using  the  ordinary 

^  Constantino:  Bioch.  Zeit.,  55,  419,  1913. 

*  Folin  and  Denis:  J.  Biol.  Client.,  11,  532,  1915. 

^The  blood  must  ht  freshly  drawn.     For  method  of  obtaining  blood,  see  page  272. 


BLOOD    ANALYSIS  279 

cylinder  for  the  standard  and  replacing  the  other  cylinder  and  prism  by  the  polari- 
scope  tube,  containing  the  unknown,  which  usually  fits  properly  in  place  in  the 
colorimeter. 

The  unknown  solution  remains  stationary  (100  mm.)  and  the  standard  is 
adjusted  until  the  colors  match.  An  iris  diaphragm,  such  as  is  used  in  microscopical 
work,  must  be  attached  to  the  side  of  the  colorimeter  holding  the  standard  to  reduce 
the  light  passing  through  the  standard  solution.  This  is  necessary  in  order  to 
obtain  two  fields  of  the  same  tint.  The  calculation  is  made  in  the  usual  manner  for 
colorimetric  determinations,  the  amount  of  ammonia  in  the  unknown  being  directly 
proportional  to  the  reading  of  the  standard  and  its  concentration. 

7.  Total  Nitrogen. — The  total  nitrogen  of  the  blood  may  be  readily  determined 
by  the  regular  Kjeldahl  method  (see  Chapter  XXVI).  One  c.c.  of  the  blood  ac- 
curately measured  is  used  in  this  method.  The  microchemical  method  of  Folin 
and  Farmer,  as  outlined  in  the  same  chapter,  may  also  be  employed.  In  this  case 
transfer  i  c.c.  of  the  well-mixed  blood  to  a  25  c.c.  flask,  make  to  the  mark  with 
distilled  water,  mix  thoroughly  and  take  i  c.c.  of  this  diluted  blood  for  the  digestion 
and  determination  as  there  given. 

8.  Sugar,  (a)  Method  of  Lewis  and  Benedict. '—Principle. — The  red 
color  obtained  by  heating  a  glucose  solution  with  picric  acid  and  sodium 
carbonate  is  employed  as  the  basis  of  the  colorimetric  determination. 
The  blood  protein  is  removed  by  precipitation  with  picric  acid. 

Procedure. — Two  c.c.  of  blood  are  aspirated  through  a  hjrpodermic  needle* 
and  a  piece  of  rubber  tubing  into  an  Ostwald  pipette,  a  little  powdered  potassium 
oxalate  in  the  tip  of  the  pipette  preventing  clotting.  The  blood  is  drawn  up  a 
Httle  above  the  mark  and  the  end  of  the  pipette  is  closed  with  the  finger.  After 
the  rubber  tubing  and  needle  are  disconnected,  the  blood  is  allowed  to  flow  back 
to  the  mark  and  is  discharged  at  once  into  a  25  c.c.  volumetric  flask  containing 

5  c.c.  of  water.  The  contents  of  the  flask  are  shaken  to  insure  thorough  mixing 
and  the  consequent  hemolysis  of  the  blood.  Then  15  c.c.  of  saturated  aqueous 
solution  of  picric  acid  are  added,  as  well  as  a  drop  or  two  of  alcohol  to  dispel  any 
foam,  and  the  contents  of  the  flask  are  made  up  to  the  mark  with  water  and  then 
shaken.  After  filtration  8  c.c.  ahquots  are  measiu-ed  out  into  large  Jena  test- 
tubes  for  duplicate  determinations.  Two  c.c.  of  saturated  picric  acid  solution  and 
exactly  i  c.c.  of  10  per  cent  sodivmi  carbonate  are  added  (as  well  as  two  glass 
beads  and  2  or  3  drops  of  mineral  oilj,  and  the  contents  of  the  tube  are  evaporated 
rapidly  over  a  direct  flame  imtil  precipitation  occurs.  About  3  c.c.  of  water  are 
added,  the  tube  is  again  heated  to  boiling  to  dissolve  the  precipitate,  the  contents 
of  the  tube  are  transferred  quantitatively  to  a  10  c.c.  volumetric  flask, ^  cooled, 
made  up  to  the  mark,  shaken,  and  then  filtered  through  cotton  into  the  chamber 
of  a  Duboscq  colorimeter  (see  Fig.  153,  p.  486).  The  color  is  compared  at  once 
with  that  obtained  from  0.64  mg.  of  glucose,  5  c.c.  of  satiu-ated  picric  acid,  and  i 
c.c.  of  19  per  cent  sodium  carbonate,  when  evaporated  to  precipitation  over  a  free 

1  Lewis  and  Benedict:  Jour.  Biol.  Chem.,  20,  61,  1915.     For  modilication  see  Myers 

6  Bailey:  Jour.  Biol.  Chem.,  24,  147,  1916. 

*  It  may  be  more  convenient  to  draw  about  5  c.c.  of  blood  directly  into  a  test-tube 
containing  a  little  finely  powdered  potassium  oxalate  and  removing  2  c.c.  portions  of  this 
with  the  Ostwald  pipette. 

*  In  case  of  hyperglycemia  the  final  volume  of  the  reaction  fluid  is  made  25  c.c.  or  50 
c.c,  and  the  results  are  accordingly  multiplied  by  2.5  or  5.0. 


28o  PHYSIOLOGICAL   CHEMISTRY 

flame  and  diluted  to  lo  c.c.  as  was  the  unknown,  or  against  the  picramic  acid 
standard  mentioned  below.  ^ 

Calculation. — If  directions  are  followed  exactly  the  calculation  is  as  follows : 

reading  of  standard 
Milligrams  glucose  m  unknown  =        ,.        *      u X  milhgrams  of  glucose 

in  standard. 

(b)  Pearce's  Modification  of  Lewis-Benedict  Method. ^ — ^This  modification 
entails  the  use  of  an  autoclave  instead  of  the  free  flame  and  has  the  advantages 
of  decreasing  danger  of  loss  and  making  it  possible  to  carry  out  a  large  number 
of  estimations  at  one  time.  Proceed  exactly  as  in  the  Lewis-Benedict,  but  use 
6  c.c.  of  the  the  picric  acid  filtrate  instead  of  8  c.c.  and  instead  of  heating  over  the 
free  flame  introduce  into  an  autoclave  for  15-30  minutes  at  about  20  pounds 
pressure  to  the  square  inch.  Compare  with  standard  in  a  colorimeter.  The 
standard  recommended  by  Lewis  and  Benedict  may  be  diluted  one-fourth  or 
allowed  for  by  calculation,  since  6  c.c.  of  filtrate  are  used  in  place  of  8  c.c. 

(c)  Micro -method  of  Bang. — Principle. — Two  or  3  drops  of  blood 
are  transferred  to  a  small  weighed  piece  of  blotting  paper  and  the 
paper  again  weighed  to  determine  the  amount  of  blood.  The  paper 
is  then  treated  with  boiling  acidified  KCl  solution  which  coagulates 
the  protein  and  allows  the  sugar  to  diffuse  out.  The  sugar  solution 
thus  obtained  is  boiled  with  alkaline  cupric  chloride  solution.  The 
amount  of  cuprous  chloride  formed  by  the  reducing  action  of  the 
sugar  is  determined  by  titration  with  standard  iodine  solution. 

Procedure. — Small  pieces  of  good  absorbent  paper,  about  16X28  mm.  in 
size,'  weighing  about  100  mg.  and  held  by  a  small  spring  cUp,  are  used.  To  one 
of  these  previously  weighed*  transfer  2-3  drops  (about  120  mg.)  of  blood  obtained 
by  piercing  the  cleansed  finger.  "Weigh  again  immediately  and  determine  by 
subtraction  the  weight  of  blood  taken. 

^  Permanent  Standard. — A  solution  of  picramic  acid  makes  a  very  satisfactory  permanent 
standard.  The  color  is  identical  in  quality  with  that  formed  in  the  method  above  and  its 
solution  keeps  perfectly.     The  formula  of  the  permanent  standard  is: 

Picramic  acid  o .  064  gram 

Sodium  carbonate  (anhydrous)  o.  100  gram 

Water  to  make  1000. o      c.c. 

Dissolve  the  picramic  acid  with  the  aid  of  heat  in  25  to  50  c.c.  of  distilled  water  which 
has  been  made  alkaUne  with  sodium  carbonate.  Cool  and  dilute  to  i  liter.  This  solu- 
tion has  the  same  intensity  of  color  as  that  obtained  by  the  proposed  method  with  0.64 
mg.  of  sugar  when  the  final  volume  of  the  reaction  fluid  is  made  10  c.c.  The  solution  shoidd 
be  standardized  against  pure  glucose. 

A  satisfactory  preparation  of  picramic  acid  may  be  obtained  from  the  J.  T.  Baker 
Chemical  Co.,  Phillipsburg,  N.  J. 

^Pearce:  Jour.  Biol.  C/iem.,  22,  525,  1915. 

*  Suitable  pieces  of  paper,  weighed,  ready  for  use,  and  with  clip  attached,  may  be  ob- 
tained from  Warmbrunn  and  Quilitz,  Berlin.  A  suitable  paper  may  also  be  obtained  from 
Griffin  and  Sons,  London,  or  Grave  of  Stockholm.  Unless  specially  prepared,  the  paper 
should  be  repeatedly  washed  with  large  volumes  of  hot  water  acidified  with  acetic  acid  to 
remove  impurities. 

*  The  weighing  is  preferably  made  on  a  special  torsion  micro-balance  which,  as  well  as 
the  other  apparatus  used  in  this  method,  may  be  obtained  from  either  of  the  firms  mentioned 
in  Note  3.  The  weighing  must  be  made  in  a  few  seconds  and  with  an  accuracy  of  about 
I  mg. 


BLOOD    ANALYSIS  28 1 

Coagulation  of  Blood  Protein. — Transfer  the  piece  of  paper  to  a  test-tube  and 
add  6.5  c.c.  of  boiling  acid-potassium  chloride  solution^  and  let  stand  half  an  hour. 
The  clear  solution  containing  the  sugar  is  poured  into  a  50  c.c.  Jena  flask  the 
flange  of  which  has  been  removed.  Wash  the  paper  and  tube  again  with  6.5  c.c. 
of  hot  salt  solution  and  transfer  washings  to  the  flask.     Cool. 

Reduction  of  Cupric  Chloride. ^ — Attach  to  the  mouth  of  the  flask  a  piece  of 
tight-fitting  rubber  tubing  about  2  inches  long  (see  Fig.  81  j,  provided  with  a 
clamp  which  permits  of  shutting  off  the  contents  of  the  flask  from  the  outside  air. 
Now  add  to  the  flask  i  c.c.  of  the  cupric  chloride  solution. ^  Heat  so  that  the 
solution  is  brought  to  a  boil  in  one  minute  and  30  seconds  ''an  error  of  five  seconds 
may  be  disregarded).  Allow  to  boil  for  exactly  two  minutes;  at  the  end  of  this 
time  tighten  the  clamp  over  the  mouth  of  the  flask.  At  the 
same  time  remove  from  the  flame  and  cool  at  once  under  the 
tap  for  about  a  minute. 

Titration  of  Cuprous  Chloride  Formed. — The  titration  is 
made  with  N/200  iodine  solution^  run  in  from  a  very  accurate 
burette  (preferably  a  2  c.c.  burette  graduated  in  1/50  c.c). 
Two  or  3  drops  of  starch  solution  (preferably  soluble  starch^; 
are  added  as  an  indicator.  During  the  titration  air  must  be 
excluded  to  prevent  re-oxidation.  This  is  done  by  running  a 
slow  stream  of  carbon  dioxide  from  a  generating  bottle  through 
a  small  tube  which  extends  nearly  to  the  bottom  of  the  flask. 
The  titration  should  be  carried  out  against  a  white  backgrovmd 
and  the  end  point  taken  when  the  blue  color  persists  for  20-30  Baxg  Rzduc- 
seconds.  tion  Flask. 

Calculation. — The  copper  and  other  solutions  used  in  the 
test  bind  about  0.12  c.c.  of  the  iodine  solution.     This  amount  must  hence  be 
subtracted  from  the  reading.     The  corrected  reading  is  then  divided  by  4  to 
obtain  the  number  of  milligrams  of  glucose  in  the  sample. 

Example. — If  0.68  c.c.  of  N/200  I  solution  were  required,    *     ' —  =  0.14 

4 
mg.  glucose  in  the  amount  of  blood  used.     If  140  mg.  of  blood  were  taken  for 

analysis  the  per  cent  of  glucose  in  the  blood  would  be  X0.14  mg.  =  0.1  per 

140 

cent  glucose. 

The  results  obtained  by  this  method  are  a  httle  higher  than  those  obtained 
by  other  reUable  methods  due  to  the  presence  of  certain  I-bindLng  substances  in 
blood.  As  these  appear  to  be  nearly  constant  in  amount  a  correction  may  be 
appUed.  To  obtain  true  values  for  glucose  of  the  blood  therefore  subtract 
0.015  per  cent  from  the  value  obtained  as  above,  o.i  per  cent  —  0.015  per  cent 
=  0.085  per  cent  glucose. 

^  Consisting  of  1360  c.c.  of  saturated  KCl  to  which  is  added  640  c.c.  of  water  and  1.5 
c.c.  of  25  per  cent  HCL. 

*  Copper  solution.  Introduce  into  a  1000  c.c.  flask  700  c.c.  of  boiled  and  cooled  water. 
Warm  to  about  3o°C.  and  add  i6o  grams  of  pure  potassium  bicarbonate  in  powder  form. 
When  dissolved  add  66  grams  of  pure  KCl.  Cool  and  then  add  100  grams  potassium  car- 
bonate. Finally  add  100  c.c.  of  4.4  per  cent  solution  of  pure  crystalline  copper  sulphate. 
Let  stand  a  short  time,  then  make  to  mark  with  boiled  water.  Allow  to  stand  a  day  or  so 
before  using. 

*  N/200  I  solution,  made  fresh  each  day.  DUute  N/io  I  solution  20  times,  or 
make  as  follows:  Introduce  into  a  100  c.c.  flask  2  grams  KI,  1-2  c.c.  of  2  per  cent  KIOj 
solution  and  5  c.c.  of  N/io  HCl.     Make   to  mark  with  boiled  and  cooled  distilled  water. 

*  A  I  per  cent  solution  of  Kahlbaum's  soluble  starch  in  a  saturated  KCl  solution. 


252  PHYSIOLOGICAL   CHEMISTRY 

To  secure  accurate  results  the  method  of  Bang  must  be  rigidly  con- 
trolled, all  new  solutions  and  absorbent  papers  being  checked  up 
against  pure  0.2  per  cent  glucose  solutions.  Taylor  and  Hulton^ 
also  suggest  the  following  precautions.  A  blank  check  must  be  made 
on  the  reagents  each  day  an  estimation  is  made.  0.10-0.15  gram  of 
blood  should  be  taken  and  must  spread  smoothly  on  the  paper.  The 
proteins  are  best  coagulated  by  heating  of  the  blood-impregnated 
papers  in  the  hot  air  oven  at  100°  (as  recommended  by  Gardner  and 
McLean)-  for  five  minutes  with  corks  of  flasks  inverted.  The  solu- 
tion should  be  boiled  four  minutes  for  complete  reduction.  The 
iodine  solution  must  be  fresh  each  day  and  checked  each  day.  Deter- 
minations should  be  made  in  triplicate.  Results  cannot  be  depended 
upon  to  be  more  accurate  than  to  0.005  gram  glucose  in  100  c.c.  blood. 
Other  authors  have  recommended  that  an  hour  instead  of  half  an  hour 
be  allowed  for  the  diffusion  of  the  blood  sugar,  the  fluid  being  brought 
to  the  boiling-point  twice  during  this  period  or  kept  in  a  bath  at  4o°C. 

Method  of  Epstein.  2 — Principle. — This  method  is  a  modification  of  the  Lewis 
and  Benedict  procedure,  being  based  on  the  same  principle  but  making  possible  the 
determination  of  reducing  sugar  in  finger  blood  (0.1-0.2  c.c.)  with  a  sufficient 
degree  of  accuracy  for  clinical  purposes,  and  with  little  expenditure  of  time.  In- 
stead of  a  Duboscq  colorimeter  the  less  expensive  Sahli-Gower  hemoglobin  color- 
imeter is  recommended. 

Procedure. — The  apparatus*  shown  in  the  illustration  (Fig.  82)  and  the  fol- 
lowing reagents  are  necessary: 

1.  Picric  acid,  saturated  solution. 

2.  Sodium  carbonate,  10  per  cent  solution. 

3.  Sodium  fluorid  or  potassium  oxalate,  2  per  cent  solution. 

Put  one  or  two  drops  of  the  fluorid  or  oxalate  solution  into  the  graduated  test- 
tube  (see  illustration).  By  means  of  the  blood  pipette,  3,  0.2  c.c.  of  blood  is 
obtained  from  the  tip  of  the  finger  or  the  lobe  of  the  ear  and  is  discharged  into  the 
tube  containing  the  fluorid  solution.  The  pipette  is  rinsed  two  or  three  times 
with  distilled  water  and  the  washings  added  to  the  blood  in  the  tube.  Distilled 
water  is  then  added  to  the  i.o  c.c.  mark.  After  laking  of  the  blood  has  taken  place, 
picric  acid  is  added  to  this  (a  few  drops  at  a  time)  up  to  the  2.5  c.c.  mark,  shaking 
the  tube  gently  with  each  addition  of  the  acid.  Precipitation  of  the  blood-proteins 
takes  place;  the  sugar,  together  with  an  excess  of  picric  acid  sufficient  for  the 

^Taylor  and  Hulton:  Jour.  Biol.  Chem.,  22,  63,  1915. 

2  Gardner  and  McLean:  Biochem.  J.,  8,  391,  1914. 

^  Epstein:  /.  Am.  Med.  Assn.,  63,  1667,  1914. 

*  The  tubes  belonging  to  this  hemoglobinometer  are  not  all  equally  calibrated.  With 
some  the  50  per  cent  mark  represents  a  volume  of  i.o  c.c;  with  others,  i.o  c.c.  of  fluid 
reaches  up  to  the  43,  45,  46  or  47  per  cent  mark.  The  error  in  the  caUbration  is  generally 
below  the  10  per  cent  mark;  the  graduations  above  this  mark  are  usually  correct.  By 
means  of  the  standard  1.0  c.c.  pipette  one  can  readily  determine  whether  or  not  a  given 
tube  is  properly  calibrated.  In  order  to  facilitate  a  direct  reading  of  the  percentage 
of  sugar  on  these  hemoglobinometer  tubes,  it  is  essential  to  have  1.0  c.c.  of  fluid  stand  at 
mark  50.  To  overcome  a  discrepancy  (if  any  exists)  in  the  calibration  of  a  given  tube, 
one  may  put  one,  two  or  three  small  glass  beads  in  the  bottom  of  the  tube,  of  such  size 
as  to  raise  the  meniscus  of  1.0  c.c.  of  fluid  up  to  the  50  per  cent  mark. 


BLOOD    ANALYSIS 


283 


reaction,  stays  in  solution.  The  tube  is  finally  shaken  vigorously  (covering  the 
end  of  the  tube  with  the  finger)  and  the  contents  filtered  through  a  small  filter,  or, 
better  still,  centrifuged  for  one  or  two  minutes. 

One  c.c.  of  the  filtrate  or  the  clear  supernatant  fluid  obtained  on  centrifugal- 
ization  is  withdrawn,  put  into  the  plain  test-tube,  and  heated  carefully  over  the 
naked  flame.  The  contents  of  the  tube  are  boiled  until  all  but  2  or  3  drops  of  the 
solution  is  evaporated.  One-half  c.c.  of  the  10  per  cent  sodium  carbonate  solution 
is  then  added  and  the  tube  heated  again  until  the  contents  are  concentrated  to 
a  small  volume  equal  to  about  2  or  3  drops.  The  color  of  the  fluid  changes  from 
yellow  to  deep  red  or  reddish  brown  and  the  reaction  is  completed. 

Three  or  4  drops  of  distilled  water  are  added  and  the  tube  warmed  gently.  The 
contents  are  then  transferred  to  the  graduated  tube  of  the  hemoglobinometer. 
The  boiling  tube  is  rinsed  several  times  with  water  (using  ordy  3  or  4  drops  at  a 
time).  The  tube  is  warmed  with  each  rinsing  before  transferring  the  contents  to 
the  graduated  tube.  The  volume  of  fluid  is  then  made  up  to  the  mark  50  on  the 
scale. 


Fig.  S2. — App.\r.a.tus  for  Epstein's  Sugar  Method. 

The  color  of  the  resulting  solution  is  compared  with  that  of  the  two  standard 
tubes,  A  and  B  which  accompany  the  instrument.  (A  solution  of  picramic  acid 
of  the  proper  strength,  prepared  as  described  on  p.  280,  may  be  used  as  a  standard.) 
If  it  is  darker  than  standard  A  (representing  0.05  per  cent  of  sugar)  and  lighter 
than  standard  B  (representing  o.i  per  cent),  the  first  standard  is  used  for  com- 
parison. In  either  case  the  solution  in  the  graduated  tube  is  diluted  gradually 
with  water  (just  as  is  usually  done  in  hemoglobin  estimations)  untU  the  colors 
match. 

The  percentage  of  sugar  in  the  blood  is  then  computed  thus:  Using  the  lighter 
standard  A  the  figure  on  the  scale,  divided  by  1000  represents  the  percentage  of 
sugar  in  the  blood.     For  example,  the  tube  reads  86;  then  the  result  is 

86 

=0.086  per  cent 

1000 

When  Standard  B  is  used  for  comparison,  the  figure  on  the  scale  is  multiplied 
by  2  and  divided  by  1000.  For  example,  the  tube  reads  73;  then  the  percentage 
of  sugar  is 

73X2 
1000 


■0.146  per  cent 


284  PHYSIOLOGICAL   CHEMISTRY 

With  the  instructions  given,  the  above  formulas  may  be  used  for  direct  com- 
putation of  the  percentage  of  sugar  only,  when  0.2  c.c.  of  blood  is  used  in  the 
determination.  When,  however,  only  o.i  c.c.  of  blood  is  used,  the  formulas  apply 
as  well,  but  the  value  obtained  must  be  multiplied  by  2. 

It  is  better,  in  cases  in  which  a  high  sugar  content  in  the  blood  is  suspected  (in 
diabetes  for  example)  to  use  only  o.i  c.c.  of  blood  for  the  determination.  In  all 
other  cases  0.2  c.c.  of  blood  should  be  used. 

8.  Acetone  Bodies^  (Acetone,  Diacetic  Acid  and  /3-Hydroxybutyric  Acid). — 
Marriott-Scott-Wilson  Method.  ^^ — (a)  Acetone  and  Diacetic  Acid. — Draw  10  c.c. 
of  blood  from  a  superficial  vein  by  a  sterile  graduated  syringe  and  run  it  into  about 
40  c.c.  of  0.5  per  cent  potassium  oxalate  solution.  Fit  up  a  Kjeldahl  distillation 
apparatus  using  an  800  c.c.  flask,  provided  with  a  dropping  funnel,  the  delivery 
tube  of  the  condenser  dipping  beneath  the  surface  of  the  water  in  a  receiving 
flask.  Introduce  into  the  Kjeldahl  flask  100  c.c.  of  water  and  i  c.c.  of  glacial 
acetic  acid.  Bring  the  acidified  water  to  a  boil  and  then  run  the  diluted  blood 
in  slowly  through  the  dropping  funnel. 

Boil  for  30  minutes  after  the  last  blood  is  run  in. '  To  the  distillate  add  a  little 
dilute  sulphuric  acid  and  redistil.  To  this  distillate  add  20  c.c.  of  hydrogen 
peroxide  solution  and  a  sUght  excess  of  alkali  and  redistil  again.  The  final  dis- 
tillate is  caught  in  small  Erlenmeyer  flasks  containing  an  excess  of  the  Scott- 
Wilson  "acetone  reagent"  which  has  been  recently  filtered.*  The  delivery  tube 
must  dip  under  the  surface  of  the  liquid.  It  is  not  necessary  to  distil  more  than 
10  minutes  to  get  off  all  the  acetone.  Allow  to  stand  for  10-15  minutes.  Filter 
through  an  asbestos  mat^  in  a  separable  bottom  Gooch  crucible.  Clear  filtrates 
are  more  readily  obtained  if  the  pores  of  the  filter  have  been  partly  closed  by 
filtering  through  it  a  suspension  of  talcum  powder  in  water.  If  the  first  portions 
of  the  filtrate  are  turbid,  refilter.  Wash  the  precipitate  with  cold  water  until 
the  washings  are  free  from  silver. 

With  the  aid  of  a  pointed  hooked  glass  rod  transfer  the  precipitate,  mat  and 
crucible  bottom  to  a  50  c.c.  beaker,  any  adhering  particles  of  precipitate  being 
washed  into  the  beaker  with  the  aid  of  about  10  c.c.  of  "acid  mixture."®  Add 
I  c.c.  of  N/5  potassium  permanganate,  cover  the  beaker  with  a  watch  glass  and 
boil  until  the  liqiud  is  colorless.  Add  more  permanganate,  a  few  drops  at  a  time, 
until  a  persistent  brown  color  is  obtained  which  does  not  disappear  on  boiling  for  a 
couple  of  minutes.  The  brown  color  is  then  discharged  by  the  addition  of  a  few 
drops  of  strong  yellow  nitric  acid.  The  greater  the  amount  of  acetone  present 
the  more  permanganate  is  required,  and  it  is  essential  to  the  accuracy  of  the 
method  that  an  excess  be  added  as  indicated  above,  otherwise  the  residts  are 
too  low. 

Cool  the  beaker  under  the  tap,  add  2  c.c.  of  saturated  ferric  alum  solution, 

'  For  nephelometric  method  see  p.  294. 

*  Scott- Wilson:  Jour,  of  Physiol..  42,  444,  1911. 
Marriott:  Jour.  Biol.  Client.,  16,  295,  1913. 

3  If  /3-oxybutyric  acid  is  to  be  determined  the  residue  in  the  Kjeldahl  flask  should  be 
kept  and  treated  as  outlined  in  the  latter  part  of  this  procedure. 

^  The  reagent  is  made  up  as  follows:  Mercuric  cyanide,  10  grams;  sodium  hydroxide, 
180  grams;  water,  1200  c.c.  The  solution  is  agitated  in  a  flask  and  400  c.c.  of  a  0.7268 
per  cent  solution  of  silver  nitrate  slowly  run  in.  At  least  30  c.c.  of  the  reagent  must  be 
taken  for  each  milligram  of  acetone  present. 

*  Filter  paper  cannot  be  used  as  the  strong  alkali  quickly  attacks  it. 

*  "Acid  mixture:"  Nitric  acid  40  parts;  sulphuric  acid,  5  parts;  water  55  parts. 


BLOOD   ANALYSIS  285 

and  run  in  from  a  burette  a  standard  solution  of  potassium  sulphocyanate  (approxi- 
mately 0.1  per  cent)  ^  until  a  very  faint  pinkish-brown  color  is  obtained  throughout 
the  solution.  The  end  point  which  consists  in  the  faintest  trace  of  color,  can  be 
detected  only  when  the  titration  is  performed  on  a  pure  white  surface.  A  con- 
trol beaker  with  i  drop  excess  of  sulphocyanate  should  be  on  hand  for  compari- 
son. A  whole  cubic  centimeter  of  sulphocyanate  may  be  run  in  after  the  end 
point  is  reached  without  very  greatly  darkening  the  shade. 

Calculation. — By  this  procedure  acetone  preformed  and  from  diacetic  acid 
are  determined  together.  The  amount  of  acetone  and  diacetic  acid  combined, 
in  terms  of  acetone,  may  then  be  calculated  by  multiplying  the  nimiber  of  cubic 
centimeters  of  the  KSCN  solution  used  by  the  equivalent  of  i  c.c.  of  this  solution 
in  acetone  as  determined  by  standardization.  ^  To  obtain  the  amount  of  acetone 
and  diacetic  acid  in  100  c.c.  of  blood  the  result  must  of  course  be  multipUed  by  10. 

(b)  Determination  of  /3-Hydroxybutyric  Acid. — The  residue  in  the  Kjeldahl 
flask  from  the  above  determination  is  used  in  the  determination  of  ^-hydroxy- 
butyric  acid.  While  still  hot,  precipitate  it  with  about  8  c.c.  of  10  per  cent  sodium 
carbonate,  boil  a  moment,  filter  on  a  Biichner  fuimel  and  wash  with  hot  water. 
To  the  clear  filtrate  add  15  c.c.  of  basic  lead  acetate  (U.S.P.)  and  10  c.c.  of  strong 
ammonia  and  make  to  definite  volvmie  (150  c.c.)  with  water.  Allow  the  precipi- 
tate to  settle  and  then  filter  off  on  a  dry,  folded  filter.  Take  an  aUquot  of  the 
clear  filtrate  (about  125  c.c.)  and  boil  it  to  expel  the  greater  part  of  the  ammonia. 
Cool  and  add  dilute  sulphuric  acid  to  precipitate  the  excess  of  lead  as  sulphate 
and  filter.  Add  10  c.c.  of  50  per  cent  sulphuric  acid  and  transfer  the  whole  to  a 
Kjeldahl  flask  provided  with  a  dropping  funnel.  The  contents  of  the  flask  are 
distilled  and  a  solution  of  potassium  bichromate  is  run  in  from  the  dropping  funnel 
at  such  a  rate  that  the  liquid  always  retains  some  yellow  color  and  the  volimie 
remains  at  about  100  c.c.  It  is  rarely  necessary  to  add  more  than  about  o.i  gram 
of  bichromate  and  an  excess  is  to  be  avoided.  Slow  distUlation  is  continued  for 
two  hours  and  about  100  c.c.  of  distillate  is  collected.  The  tip  of  the  deUvery  tube 
must  always  remain  under  the  surface  of  the  water  in  the  receiving  flask.  Add 
20  c.c.  of  hydrogen  peroxide,  make  sUghtly  alkaline  with  NaOH  and  distil  again. 
Catch  the  distillate  in  small  Erlenmeyer  flasks  containing  an  excess  of  the  Scott- 
Wilson  acetone  reagent  (at  least  30  c.c.  for  each  milligram  of  acetone)  and  de- 
termine the  acetone  according  to  the  procedure  outlined  in  the  preceding  method 
for  acetone  and  diacetic  acid.  Calculate  the  /3-hydroxybutyric  acid  in  terms  of 
acetone  and  express  as  miUigrams  of  acetone  per  100  c.c.  of  blood. 

9.  Cholesterol.  Method  of  Autenrieth  and  Funk} — Principle. — 
The  blood  or  serum  is  boiled  with  strong  alkali  to  saponify  the  fats. 
The  alkaline  solution  is  extracted  with  chloroform.  The  chloroform 
is  dried  and  clarified  by  means  of  anhydrous  sodium  sulphate  and 
filtration  and  then  treated  with  sulphuric  acid  and  acetic  anhydride, 
and  the  characteristic  color  reaction  of  the  Liebermann-Burchard  test 

'  The  sulphocj^anate  solution  should  be  standardized  against  pure  acetone  treated 
in  the  same  manner  as  ihe  final  tlistillate  above.  According  to  Scott-Wilson  it  may  also 
be  standardized  against  a  pure  solution  of  mercuric  nitrate  and  the  equivalent  of  i  c.c.  of 
the  KSCN  solution  in  milligrams  of  Hg  determined.  According  to  this  author  i  mg.  of  Hg 
is  equivalent  to  0.058  mg.  of  acetone. 

^Autenrieth  and  Funk:  Miinch.  mcd.  Woch.,()o,  1243,  1913.  A  slight  modification 
has  been  suggested  by  Bloor  {Jour.  Biol.  Chem.,  23,  317,  1915). 


286  PHYSIOLOGICAL    CHEMISTRY 

for  cholesterol  obtained.     The  color  is  compared  with  a  standard  in  a 
colorimeter. 

Procedure. — ^With  an  accurate  pipette  transfer  2  c.c.  of  whole  blood  or  serum 
to  a  100  c.c.  Erlenmeyer  flask,  and  add  20  c.c.  of  a  25  per  cent  potassium  hydroxide 
solution.  Heat  on  the  water-bath  for  two  hoiirs  shaking  frequently  and  adding  a 
Uttle  water  if  necessary  to  keep  from  going  to  dryness.  Pour  the  undiluted 
mixture  into  a  separatory  fixnnel  and  add  25-30  c.c.  of  chloroform.  Shake 
vigorously  for  five  minutes  and  separate.  Shake  out  with  four  more  portions  of 
20  c.c.  each  of  chloroform.  The  combined  chloroform  extracts  are  turbid  and 
of  a  green  or  brown  color  and  are  clarified  by  shaking  with  5-10  grams  of  anhy- 
drous sodium  sulphate  and  filtering.  Dilute  the  filtrate  to  100  c.c.  with  chloroform. 
Transfer  5  c.c.  of  this  extract  to  a  small  glass-stoppered  bottle  of  about  10  c.c. 
capacity,  add  2  c.c.  of  acetic  anhydride  and  o.i  c.c.  of  concentrated  sulphuric  acid 
and  shake.  Place  in  a  water-bath  at  32-35°C.  and  keep  in  the  dark  15  minutes. 
A  green  color  is  developed.  At  the  same  time  a  series  of  standards  are  prepared 
and  treated  in  the  same  maimer.  The  color  of  the  test  is  compared  with  that 
of  the  most  similar  standard  in  a  colorimeter  (see  Fig.  153,  p.  486)  and  its  color 
strength  determined. 

Five  standards  are  kept,  these  being  prepared  by  dissolving  in  100  c.c.  por- 
tions of  chloroform:  (i)  3.2;  (2)  4.8;  (3)  6.4;  (4)  8.0;  (5)  9.6  mg.,  respectively  of 
piire  cholesterol.  In  preparing  the  standards  for  comparison  5  c.c.  portions  of 
each  of  the  above  solutions  are  taken,  placed  in  small  glass-stoppered  bottles 
and  treated  as  were  the  unknowns.  Each  standard  then  represents  a  concen- 
tration of:  160  mg. ;  240  mg. ;  320  mg. ;  400  mg. ;  and  480  mg.  respectively  of 
cholesterol  in  100  c.c.  of  the  original  blood  or  sermn. 

10.  Chlorides. — Method  of  McLean  and  Van  Slyke.^ — The  determination  re- 
quires two  steps:    (i)  removal  of  proteins  and  (2)  titration  of  chlorides. 

Coagulation  of  Proteins. — Removal  of  the  proteins  may  be  accomplished  in  two 
ways,  by  coagulation  or  by  ignition.  Results  are  identical  by  both  methods,  but 
coagulation  is  the  simpler.  For  coagulation  2  c.c.  of  oxalated  plasma  (i  c.c.  may 
be  used  if  material  is  limited)  are  drawn  into  a  2  c.c.  pipette  which  has  been  cali- 
brated to  contain  2  +  0.005  c.c.  From  the  pipette  the  plasma  is  run  into  a  20  c.c. 
stoppered  volumetric  flask  which  contains  10  c.c.  of  a  10  per  cent  magnesium  sul- 
phate solution.  The  pipette  is  rinsed  twice  by  drawing  up  into  it  the  solution  from 
the  flask.  Two  drops  of  50  per  cent  acetic  acid  are  added,  the  flask  is  filled  to  the 
mark  with  water,  the  contents  are  mixed  by  inverting  the  flask,  and  heated  in  a 
bath  to  100°  for  ten  minutes.  By  keeping  the  stopper  loosely  in  place  evaporation 
is  prevented,  and  when  cool  the  contents  return  to  their  original  volume.  Ten 
minutes  on  the  steam  bath  are  sufiicient  to  coagulate  the  albumin  and  to  distribute 
the  chlorides  evenly  between  the  fluid  and  precipitated  albumin.  The  flask  is  then 
allowed  to  cool,  and  the  contents  are  poured  upon  about  0.3  gram  of  blood  char- 
coal^ in  a  small  beaker,  and  mixed.  After  a  few  minutes  the  liquid  is  filtered 
through  a  dry  folded  filter,  and  a  water-clear  filtrate  obtained. 

Titration  of  the  Protein-free  Filtrate. — In  brief,  the  chlorides  are  precipitated  in 
the  presence  of  nitric  acid  by  standard  silver  nitrate  solution,  the  silver  chloride  is 
removed  by  filtration,  and  the  excess  silver  titrated  with  standard  potassium 

^  McLean  and  Van  Slyke:  Jour.  Biol.  Chem.,  21,  361,  1915. 

^Merck's  "Blood  Charcoal  Reagent,"  purified  by  acid  and  freefr  cm  chloride,  is  used. 
No  other  form  of  charcoal  has  been  found  to  be  of  serv'ice. 


BLOOD   ANALYSIS  287 

iodide.'  The  titration  is  performed  in  the  presence  of  nitrous  acid  and  starch,  so 
that  the  first  drop  of  iodide  in  excess  of  the  silver  present  is  changed  to  free  iodine 
and  gives  the  blue  starch-iodine  color.  The  optimum  acidity  for  the  end  point  is 
fixed  by  the  addition  of  trisodium  citrate  in  amount  equivalent  (J^  mol.)  to  the 
free  nitric  acid  present.  Under  these  conditions  i  drop  of  excess  N/50  iodide 
gives  a  color  preceptible  in  150  c.c.  of  solution. 

In  detail,  the  titration  of  the  protein-free  plasma  filtrate  is  carried  out  as 
follows:  Either  10  c.c.  of  the  filtrate,  containing  the  chlorides  of  i  c.c.  of  plasma, 
are  taken  in  a  pipette  for  titration  or  the  filtrate  is  collected  directly  in  a  certified 
25  c.c.  graduated  cylinder,  where  it  is  measured,  so  that  the  entire  amount  may 
be  taken  for  titration.  In  this  way  13  to  14  c.c.  of  filtrate,  corresponding  to  1.3 
to  1.4  c.c.  of  plasma,  may  be  obtained  for  titration. 

After  either  measuring  10  c.c.  of  the  filtrate  into  a  25  c.c.  volumetric  flask  or 
recording  the  amount  of  filtrate  obtained  in  the  cylinder,  5  c.c.  of  the  acidified 
Af/29.25  silver  nitrate  solution  (Solution  I)  are  added,  and  the  whole  is  made  to  the 
25  c.c.  mark  with  water.  This  will  precipitate  up  to  10  mg.  of  XaCl.  In  samples 
with  high  percentage  of  chloride,  only  enough  filtrate  is  taken  to  keep  within  this 
limit  of  ID  mg.  Two  drops  of  octyl  (caprylic)  alcohol  are  added,  and  the  vessel  is 
stoppered  and  shaken  gently  by  inverting  it  several  times.  Immediate  coagulation 
of  the  silver  chloride  occurs.  After  allowing  five  minutes  for  complete  precipitation 
to  occur,  the  solution  is  filtered  through  a  dry  folded  filter,  and  a  perfectly  clear  and 
colorless  fiJtrate  again  obtained.  An  aliquot  part  of  the  filtrate  (20  c.c.)  is  now 
taken  with  a  pipette  for  titration. 

Just  before  titration  with  the  potassium  iodide  solution  (Solution  II)  one  adds 
a  volume  of  the  citrate  solution  (Solution  III)  equal  to  the  volume  of  Solution  I 
represented  in  the  filtrate  to  be  titrated.  If,  as  is  usually  the  case,  one  has  used 
5  c.c.  of  Solution  I  diluted  to  25  c.c,  and  taken  20  c.c.  of  the  filtrate  for  titration 
one  has  the  equivalent  of  4  c.c.  of  Solution  I  present,  and  must  accordingly  add 

^  The  following  solutions  are  required; 

I.  An  acid  Af/29.25  solution  of  silver  nitrate,  i  c.c.  of  which  is  equivalent  to  2  mg.  of 
NaCL. 

AgNOs 5.812  grams 

HNO3  (sp.  gr.  1.42) 250  c.c. 

Water  to 1000  c.c. 

II.  A  solution  of  ilf/58.5  potassium  iodide,  i  c.c.  of  which  is  equivalent  to  i  mg.  of 
NaCL. 

KI 3.0      grams 

Water  to 1000  c.c. 

This  solution  is  standardized  against  the  silver  solution  by  adding  5  c.c.  of  the  latter 
to  5  c.c.  of  Solution  III,  and  titrating  with  the  iodide  solution  to  the  blue  end-point.  The 
iodide  solution  is  then  diluted  to  such  a  degree  that  10  c.c.  are  exactly  equivalent  to  5  c.c. 
of  the  silver  solution. 

III.  A  solution,  for  use  in  the  final  titration,  containing  sodium  citrate,  sodium  nitrate, 
and  starch,  which  substances  respectively  regulate  the  acidity,  provide  an  o.xidizing  agent 
for  the  iodide,  and  serve  as  indicator. 

Sodium  citrate  (NasCsHeOT+S  1/2H2O) 446  grams 

Sodium  nitrite 20  grams 

Soluble  starch 2.5  grams 

Water  to looo  c.c. 

The  starch  is  first  dissolved  with  the  aid  of  hoat  in  about  500  c.c.  of  water.  The  citrate 
and  nitrite  are  then  added,  and  the  mixture  is  heated  until  all  is  dissolved.  The  solution, 
while  still  hot  is  filtered  through  cotton,  the  filter  washed  with  hot  water,  the  tiltrate 
allowed  to  cool,  and  made  up  to  1000  c.c.  Filtration  removes  insoluble  substances  occur- 
ring chiefly  in  the  nitrite,  and  cotton  filters  more  rapidly  than  filter  paper.  The  solution 
keeps  indefinitely.     It  becomes  cloudy  on  standing,  but  its  efficacy  is  not  impaired. 


288  PHYSIOLOGICAL   CHEMISTRY 

4  c.c.  of  Solution  III.  On  adding  Solution  III  a  slight  turbidity  appears,  which  in 
no  way  interferes  with  the  end  point. 

The  potassium  iodide  solution  is  then  run  in  from  a  burette  until  the  blue  end 
point  appears.  The  first  definite  blue  color  is  taken  as  the  end  point,  and  with 
slight  practice  is  unmistakable.  An  additional  drop,  which  may  be  used  as  a  control, 
causes  such  a  deep  blue  color  that  it  is  impossible  to  make  an  error  of  more  than  one 
drop  in  titration.  Should  the  end  point  be  accidentally  passed,  one  may  add  i  c.c. 
of  Solution  I,  I  c.c.  of  Solution  III  and  retitrate,  allowing  in  the  calculation  for  the 
extra  sUver  nitrate. 

The  result  may  be  calculated  from  the  following  formula,  which  applies  only 
when  20  c.c.  of  the  filtrate  from  silver  chloride  are  titrated: 

^  ^^  ^,        ,.  12.5  (8  —  c.c.  KI  solution  used) 

Grams  NaCl  per  liter  =  r^ — t~zti — I — ^~^ ' 

*^  c.c.  blood  filtrate  taken 

Thus,  if  13  c.c.  of  filtrate  are  obtained  and  titrated  after  precipitation  of  plasma 
protein,  and  1.60  c.c.  of  KI  are  used  in  the  final  titration, 

n           -KT  ^^         r.           ^^-SCS  -  1.60) 
Grams  JNaCl  per  liter  = =  6. 1=5. 

In  case  only  i  c.c.  of  plasma  instead  of  2  c.c.  has  been  used,  the  factor  25  replaces 
12.5  in  the  numerator.     The  error  should  be  within  a  limit  of  i  per  cent.^ 

II.  Total  Solids. — The  total  solids  of  the  blood  are  most  readily  determined  by 
using  a  type  of  weighing  bottle  differing  from  the  usual  form  merely  in  having  a 
glass  loop  attached  to  the  under  side  of  the  stopper. ^  A  block  of  filter  paper  is 
suspended  from  this  loop  by  means  of  a  small  wire  hook.  The  bottle  and  filter 
block  are  dried  and  weighed.  From  a  small  pipette  0.3-0.6  gram  of  the  well-mixed 
blood  is  allowed  to  flow  rapidly  upon  the  filter  block.  The  stopper  is  quickly 
inserted  and  the  bottle  weighed.  The  stopper  is  then  tilted  and  the  bottle  placed 
in  the  drying  oven  at  105°  over  night.  When  convenient  the  bottle  is  cooled  and 
again  weighed.     The  total  solids  are  calculated  from  the  loss  of  moisture. 

12.  Relative  Hydrogen  Ion  Concentration  of  the  Blood.  Method 
of  Levy,  Rowntree,  and  Marriott.^ — Principle. — The  blood  is  dialyzed 
against  normal  salt  solution  and  the  H  ion  concentration  of  the  protein- 
free  dialyzate  is  determined  by  the  indicator  method,  using  phenol- 
sulphonephthalein. 

Procedure. — One  to  3  c.c.  of  clear  serum  or  of  blood  is  run,  by  means  of  a 
blunt-pointed  pipette,  into  a  dialyzing  sac*  which  has  been  washed  outside  and 

1  The  exactness  of  the  method  depends  directly  on  the  care  with  which  measurements 
are  made  and  on  the  purity  of  the  reagents  used.  All  reagents  must,  of  course,  be  free 
from  substances  precipitated  by  silver,  and  are  easily  tested.  As  the  volumes  measured 
are  not  large,  however,  small  absolute  errors  in  measurement  may  cause  considerable 
percentage  error  in  the  final  result.  All  glassware  must  therefore  be  carefully  calibrated 
by  either  the  weight  of  water  contained  (flasks,  cylinders,  2  c.c.  pipettes)  or  the  weight  de- 
livered (other  pipettes,  burettes),  and  burettes  should  be  used  which  are  capable  of  being 
read  to  0.02  c.c.  and  which  deliver  small  drops.  If  these  precautions  are  followed,  one 
should  uniformly  obtain  results,  which  are  weU  within  a  limit  of  error  of  i  per  cent. 

^  Myers  and  Fine:  "Chemical  Composition  of  the  Blood  in  Health  and  Disease," 
191 5.     The  weighing  bottles  may  be  obtained  from  Eimer  and  Amend,  N.  Y. 

^Levy,  Rowntree  and  Marriott,  Arch.  Int.  Med.,  16,  389,  1915. 

*  Preparation  of  Sacs. — One  ounce  of  celloidin  is  dissolved  in  500  c.c.  of  a  mixture  of 
equal  quantities  of  ether  and  ethyl  alcohol.     The  solids  swells  up  and  disolves  with  oc- 


BLOOD    ANALYSIS 


289 


inside  with  salt  solution.'  The  sac  is  lowered  into  a  small  test-tube  fiooXio 
mm.,  inside  measurements),  containing  3  c.c.  of  salt  solution,  until  the  fluid  on  the 
outside  of  the  sac  is  as  high  as  on  the  inside.  From  5-10  minutes  are  allowed  for 
dialysis.  The  collodion  sac  is  removed  and  5  drops  of  the  indicator  (o.oi  per 
cent  solution  of  phenolsulphonephthalein )  are  thoroughly  mixed  with  the  dialyzate. 
The  tube  is  then  compared  with  the  standards-  until  the  corresponding  color  is 
found,  which  indicates  the  hydrogen  ion  concentration  present  in  the  dialyzate. 
Readings  should  be  made  immediately  against  a  white  background.  Results 
are  expressed  in  logarithmic  notation. 

Oxalated  blood  from  normal  individuals  gives  a  dialyzate  with  a  Ph  varying 
from  7.4  to  7.6,  while  that  of  serum  ranges  from  7.6  to  7.8.  In  cUnical  acidosis  fig- 
ures from  7.55  to  7.2  have  been  noted  by  this  method  for  serum  and  for  oxalated 
blood  from  7.3  to  7.1.  A  rise  in  the  H  ion  concentration  of  the  blood  is  significant 
because  it  indicates  a  failure  on  the  part  of  the  protective  mechanism  of  the  body 
to  preserve  the  proper  reaction. 


casional  gentle  shakings,  in  48  hours.  As  a  small  amount  of  brown  sediment  separates 
out  at  first,  the  solution  should  stand  for  at  least  three  or  four  days,  after  which  the  clear 
supernatant  solution  is  ready  for  use.  A  small  test-tube  (120  by  9  mm.,  inside  measure- 
ment) is  filled  with  this  mi.xture,  inverted,  and  half  the  contents  poured  out.  The  tube  is 
then  righted,  and  the  collodion  allowed  to  fill  the  lower  half  again.  A  second  time  it  is 
inverted  and  rotated  on  its  axis,  the  collodion  being  drained  off.  Care  must  be  taken  to 
rotate  the  tube,  in  order  to  secure  a  uniform  thickness  throughout.  The  tube  is  clamped 
in  the  inverted  position  and  allowed  to  stand  for  ten  minutes,  until  the  odor  of  ether  finally 
disappears.  It  is  filled  five  or  si.x  times  with  cold  water,  or  it  is  allowed  to  soak  five  minutes 
in  cold  water.  A  knife  blade  is  run  around  the  upper  rim,  so  as  to  loosen  the  sac  from  the 
rim  of  the  test-tube,  and  a  few  cubic  centimeters  of  water  are  run  down  between  the  sac 
and  the  glass  tube.  By  gentle  pulling  the  tube  is  e.xtracted,  after  which  it  is  preserved  by 
complete  immersion  in  water. 

^  The  Salt  Solution. — The  blood  or  serum  is  dialyzed  against  an  0.8  per  cent  sodium 
chloride  solution. 

Before  applying  the  test,  it  is  necessary  to  ascertain  that  the  solution  is  free  from  acids 
other  than  carbonic.  To  determine  this,  a  few  cubic  centimeters  of  the  salt  solution  are 
placed  in  a  Jena  test-tube  and  i  or  2  drops  of  the  indicator  added,  whereupon  a  yellow  color 
appears.  On  boiling,  carbon  dio.xide  is  e.xpelled,  and  the  solution  loses  its  lemon  color 
and  takes  on  a  slightly  brownish  tint.  In  the  absence  of  this  change  other  acids  are 
present,  and  the  salt  solution  is  therefore  not  suitable.  If,  on  the  other  hand,  on  adding 
the  indicator  pink  at  once  appears,  the  solution  is  alkaline  and  hence  cannot  be  used. 

-  Preparation  of  Standard  Colors. — Standard  phosphate  mi.xtures  are  prepared  according 
to  Sorensen's  directions  as  follows: 

J^5  mol.  acid  or  primary  potassium  phosphate.  9.07S  grams  of  the  pure  recrj-stallized 
salt  (KH2PO4)  is  dissolved  in  freshly  distilled  water  and  made  up  to  i  liter. 

Hs  mol.  alkaline  or  secondary  sodium  phosphate.  The  pure  recrystallized  salt 
(Xa2HP04.i2HoO)  is  exposed  to  the  air  for  from  ten  days  to  two  weeks,  protected  from 
dust.  Ten  molecules  of  water  of  crystallization  are  given  off  and  a  salt  of  the  formula 
Na3HP04.2H20  is  obtained.  11.876  grams  of  this  is  dissolved  in  freshly  distilled  water 
and  made  up  to  i  liter.  The  solution  should  give  a  deep  rose-red  color  with  phenol- 
phthalein.     If  only  a  faint  pink  color  is  obtained,  the  salt  is  not  sufficiently  pure. 

The  solutions  are  mixed  in  the  proportions  indicated  below  to  obtain  the  desired  Pii. 

TABLE  FOR  PREPARATION  OF  STANDARD  COLORS 


Ph 

6.46.6 

6.87.0I7.1 

7.2 

7-3 

7.4 

7-5 

7.6 

7-7 

7.81  8.0 

8.2 

8.4 

Primary        potassium 
phosphate  c.c 

73 

63 

51    37    32 

27 

23 

19 

IS. 8 

13-2 

II. 0 

8.8;  5-6 

3.2 

2.0 

Secondary          sodium 
phosphate  c.c. ..... 

27 

37 

49    63    68 

73 

77 

81 

84.2 

86.8 

89.091.2  94.4 

96.8 

98.0 

19 


290  PHYSIOLOGICAL   CHEMISTRY 

13.  The  Determination  of  Epinephrin  (Adrenalin)  in  the  Adrenal 
Glands.^ — Principle. — It  has  not  yet  been  found  possible  to  determine 
adrenalin  in  the  blood  by  chemical  methods.  In  the  adrenals  it  may  be 
determined  by  the  method  given  below. 

The  method  is  based  upon  the  quantitative  production  of  blue 
color  when  a  solution  of  adrenalin  is  treated  with  phosphotungstic 
acid.  Uric  acid  and  other  substances  likewise  give  the  reaction  but 
can  be  disregarded  when  the  determination  is  made  upon  adrenal 
glands. 

Procedvire. — Rub  the  weighed  gland  thoroughly  in  a  mortar  with  fine  sand 
and  N/io  hydrochloric  acid.  The  mixture  is  then  rinsed  into  an  Erlenmeyer 
flask  with  more  N/io  acid  and  water,  using  in  all  about  15  c.c.  of  the  acid  for  each 
2  grams  of  gland  and  about  three  times  as  much  water.  Heat  to  boiling  to  dis- 
solve the  epinephrine.  Then  add  5  c.c.  of  10  per  cent  sodium  acetate  solution  for 
each  15  c.c.  of  hydrochloric  acid  present  and  heat  again  to  boiling.  Transfer 
the  whole  mixture  (except  the  sand)  to  a  volumetric  flask  (capacity  100  c.c.  for 
each  2  grams  of  gland)  and  make  to  mark  with  water.  Filter.  Pipette  5  c.c.  of 
the  clear  extract  into  a  100  c.c.  measuring  flask  and  i  c.c.  of  a  fresh  uric  acid 
solution  containing  i  mg.  of  uric  acid  into  another  100  c.c.  flask.  To  each  flask 
add  2  c.c.  of  uric  acid  reagent  (see  Folin  and  Denis'  method  for  uric  acid, 
page  274)  and  20  c.c.  of  saturated  sodiiom  carbonate  solution.  Allow  to  stand 
for  two  to  three  minutes,  dilute  to  the  mark,  shake  thoroughly,  and  compare 
in  the  usual  maimer  in  a  Duboscq  colorimeter  with  the  uric  acid  standard  set 
at  20  mm.  Calculate  as  if  the  solution  contained  uric  acid,  and  divide  the 
result  by  three  to  obtain  the  equivalent  in  epinephrine  which  gives  three  times  as 
much  color  as  an  equal  weight  of  uric  acid. 

Nephelometric  Methods 

The  Nephelometer. — The  nephelometer  is  an  instrument  for 
measuring  the  density  of  precipitates  and  thus  determining  the  amount 
of  any  substance  which  can  be  obtained  in  the  form  of  a  suitable 
suspension.  It  is  somewhat  similar  in  form  and  principle  to  a  color- 
imeter. It  differs  from  the  latter  in  that  the  light  which  reaches  the 
eye  is  not  transmitted  light,  which,  on  the  contrary,  is  excluded,  but 
light  reflected  from  the  particles  of  the  suspension.  The  brightness 
of  the  two  fields  is  compared  instead  of  their  colors.  It  is  adapted 
particularly  for  the  determination  of  substances  that  in  very  dilute 
solution  may  be  precipitated  in  the  form  of  suspensions  which  do  not 
agglutinate  appreciably  in  the  time  required  for  making  readings 
(10-20  minutes).  The  method  has  been  adapted  to  the  determination 
of  proteins  in  digestion  mixtures,  milk,  urine,  etc.;^  nucleic  acids  ;^ 

1  Folin,  Cannon,  and  Denis:  Jour.  Biol.  Chem.,  13,  477,  1913. 

'^  Kober:  Jotir.  Biol.  Chem.,  13,  485,  1913;  Jour.  Am.  Ch.  Soc,  35,  1585,  1913;  Folin 
and  Denis:  Jour.  Biol.  Chem.,  18,  273,  1914. 

2  Kober  and  Graves:  Jour.  Am.  Chem.  Soc,  36,  1304,  1914. 


BLOOD   ANALYSIS 


291 


chlorides/  phosphates,  and  phosphatides  in  blood,  etc.;-  fats  in  milk, 
blood,  etc.;^  acetone  bodies  in  urine  and  blood ;^  uric  acid  and  purine 
bases ;^  ammonia;^  calcium;^  silver,  etc.,  and  is  continually  finding 
new  applications.  It  is  possible  to  determine  very  minute  amounts 
of  substances,  entirely  outside  of  the  range  of  gravimetric  methods  of 
analysis,  and  hence  the  procedure  may  be  used  where  the  amount  of 
material  is  very  limited.  If  properly  carried  out  the  limits  of  error 
of  the  method  are  not  greater  than  those 
of  the  colorimetric  methods  commonly 
used.  Below  will  be  found  descriptions 
of  and  figures  representing  two  satisfac- 
tory types  of  nephelometer. 

The  Duboscq  colorimeter  has  been 
adapted  for  nephelometric  purposes  by 
Kober^  and  by  Bloor.^  Bloor's  nephelom- 
eter is  illustrated  in  Figs.  83  and  84.  The 
brass  plate  carrying  the  colorimeter 
plungers  is  replaced  by  the  plate  A  with 
two  slots  in  which  are  supported  the 
nephelometer  tubes  B  with  their  flanges 
resting  on  the  edges  of  the  slots.  The  slots 
are  so  cut  that  the  center  fines  of  the  tubes 
are  exactly  in  line  with  the  centers  of  the 
lower  openings  of  the  prism  case  E.     If 

desired  they  may  be  countersunk  to  re-  fig.Ss-bI^ok'sNep^lometer. 
ceive  the  flanges.     The  colorimeter  cups 

are  replaced  by  the  jackets  C  which  project  through  the  holes  in  the 
cup  supports  F  and  are  supported  on  them  by  the  collars  D.  They 
move  when  the  cup  supports  move.  The  mirror  is  turned  to  the 
horizontal  position  so  that  it  reflects  no  light.  The  fight  in  the  nephe- 
lometer comes  from  in  front  and  not  from  below  (see  Fig.  84).  The 
nephelometer  tubes  are  small  test-tubes  100X15  mm.,  preferably 
made  from  the  same  sample  of  colorless  glass  tubing  so  that  they  are 
of  exactly  the  same  bore.  The  flanges  at  the  top  should  be  well  made 
so  that  the  tubes  rest  firmly  and  evenly  in  the  slots.     The  glass  should 


'  Richards:  Zeilschr.  f.  anorg.  CItem.,  7,  269,  1895. 

-  Greenwald:  Jour.  Biol.  Cliem.,  21,  29,  1915;  Bloor:  Jour.  Biol.  Chem.,  22,  133,  1915; 

Kober  and  Egerer:  Jour.  Am.  Chem.  Sac,  37,  2373,  1915. 
^  Bloor:  Jour.  Biol.  Client.,  17,  377,   1914;  /.  Am.  Chem.  Soc,  36,  1300,  1914. 
■•  Folin  and  Denis:  Jour.  Biol.  Chem.,  18,  263,  1914;  Marriott:  same,  16,  289,  1913. 

*  Graves  and  Kober:  Jour.  Avi.  Chem.  Soc,  37,  2430,  1915. 
"  Graves:  /.  Am-.  Chem.  Soc.,  37,  1181,  1915. 

'Lyman:  Jour.  Biol.  Chem.,  21,  551,  1915. 

*  Kober:  Jour.  Biol.  Chem.,  13,  485,  1913;  Jour.  Am.  Chem.  Soc.,  35,  15S5,  1913. 
8  Bloor:  Jour.  Biol.  Chem.,  22,  145,  1915. 


292 


PHYSIOLOGICAL   CHEMISTRY 


be  as  free  as  possible  from  imperfections  and  striations.  After  the 
tubes  are  made  and  fitted  into  place  the  jackets  are  moved  up  on 
each  tube  by  means  of  the  rack  and  pinion  until  the  indicator  on  the 
scale  is  exactly  at  zero.  Marks  are  made  on  each  tube  at  the  point 
reached  by  the  top  of  the  jacket  and  the  portion  of  the  tube  above  that 
point  is  made  opaque  by  a  ring  Bl  of  black  paper  or  paint.  Tubes 
and  jackets  are  then  marked  right  and  left  and  always  used  on  the 
same  side.  Since  it  is  rare  to  find  two  tubes  which  when  filled  with  the 
same  solution  give  exactly  the  same  readings  it  is  necessary  to  take  this 
fact  into  account  and  correct  accordingly. 

The  jackets  C  are  made  of  tubing  (metal  or  glass)  a  little  larger 
than  the  tubes  and  about  the  same  length  (they  should  clear  the 
mirror  when  it  is  turned  horizontal),  closed  at  the  bottom  and  made 


Path  of  Light 


Partition 


Curtain 


Fig.  84. — Nephelometer  in  Position,  Showing  Relation  to  Source  of  Light. 


light  tight  by  black  paint  or  paper.  The  collars  D  supporting  the 
jackets  may  be  made  of  cork  or  more  permanently  of  metal.  A  little 
cotton  wool  in  the  bottom  of  the  jackets  will  prevent  breakage  if  the 
tubes  should  fall  into  the  jackets. 

The  openings  in  the  prism  case,  particularly  the  lower  ones,  should 
be  protected  against  accidental  splashing  by  thin  glass  plates  (thick 
cover  slips)  which  are  held  in  place  by  a  little  glue. 

Artificial  light  is  necessary  and  the  lamp  should  be  enclosed  in 
a  tight  box  into  one  end  of  which  the  nephelometer  fits  snugly.  A 
partition  extending  part  way  up  the  box  as  shown  in  the  diagram 
(Fig.  84)  serves  the  double  purpose  of  shutting  off  the  light  from 
the  lower  part  of  the  instrument  and  of  providing  a  stop  against 
which  the  instrument  is  pushed,  so  that  its  distance  from  the  light 


BLOOD    ANALYSIS 


293 


is  kept  constant.  The  box  is  conveniently  made  without  a  bottom 
and  the  end  closed  with  a  dark  curtain  after  the  nephelometer  is 
pushed  into  place.  The  inside  of  the  box  should  be  painted  black. 
A  dark  room  is  desirable  but  not  necessary,  as  the  instrument  may 
be  used  satisfactorily  in  a  room  darkened  by  a  dark  shade  or  even  in  a 
dark  corner  of  the  laboratory. 

The  relations  of  the  nephelometer  and  the  light  source  may  be 
seen  in  the  diagram,  Fig.  84.  The  lamp  used  is  an  ordinary  50- 
watt  tungsten  ("Mazda")  supported  by  a  bracket  about  30  cm.  from 
the  nephelometer  and  at  the  height  of  the  nephelometer  tubes.  The 
change  from  one  instrument  to  the  other  can  be  made  in  one  or  two 


Fig.  85. — Lenzmaxn-Kober  Nephelometer. 

minutes,  since  it  consists  essentially  only  in  unscrewing  the  brass 
plate  carrying  the  plungers  and  screwing  on  the  plate  to  carry  the 
nephelometer  tubes.  The  extra  parts  needed,  plate,  tubes,  and 
jackets,  are  few  and  can  be  made  if  necessary  from  material  at  hand 
in  any  laboratory  and  by  anyone  with  a  sUght  degree  of  mechanical 
skill. 1 

The  above  description  applies  t)nly  to  the  later  type  of  colorime- 
ter where  the  cups  move  and  the  prisms  are  stationary.  The  changes 
required  to  convert  the  older  type  of  instrument  are  more  complicated 
and  scarcely  to  be  advised  unless  the  instrument  is  to  have  fairly 

*  The  extra  parts  necessary  for  the  conversion  of  the  colorimeter  into  the  nephelometer 
may  be  obtained  from  the  International  Instrument  Co.  of  Cambridge,  Mass. 


294  PHYSIOLOGICAL   CHEMISTRY 

continuous  use  as  a  nephelometer.  If  the  change  is  desired  the  nephe- 
lometer  tubes  are  to  be  supported  in  the  same  way  as  above,  but  the 
jackets  must  be  carried  on  special  brackets  which  are  made  to  replace 
the  brackets  carrying  the  plungers.  The  nephelometer  tubes  must  be 
stationary,  the  jackets  being  the  movable  parts. 

Kober  has  devised^  a  combined  colorimeter  and  nephelometer  less 
expensive  than  the  Duboscq  apparatus  and  which  may  be  obtained 
in  this  country.^  A  diagram  illustrating  the  construction  of  this 
Lenzmann-Kober  nephelometer  is  given  in  Fig.  85,  page  293. 

Nephelometric  Calculations. — The  amounts  of  precipitate  in  solu- 
tions examined  nephelometrically  is  not  exactly  inversely  proportional 
to  the  readings  of  the  scale.  When  the  concentration  of  the  unknown 
and  of  the  standard  are  within  10  per  cent  of  each  other  (or  within 
about  20  per  cent  if  the  readings  are  made  at  depths  as  great  as  50- 
60  mm.)  accurate  results  may  however  be  obtained  directly.  If  the 
variations  are  greater  than  this  a  correction  is  necessary.  Kober^ 
has  proposed  an  equation  to  supply  this  correction  and  thus  make 
possible  very  accurate  work  under  conditions  of  moderate  variations 
of  concentration.     The  equation  is  as  follows: 

5      {l—x)sk 


y  = 
X 


or 


s-\-sk-\-\/{s+sky  —  ^sky 

X  = 

2y 

where  y  =  height  of  unknown  solution,  on  the  left  side  of  the  instru- 
ment, when  standard  solution  is  kept  on  the  right  side  at  a  definite 
height,  s  =  height  of  standard  solution  on  the  left  side  and  x  =  the 
ratio  of  the  concentrations  of  the  two  solutions. 

k  =  where  K  =  a,  constant,  obtained  by  substitution  of  standardi- 
zation values  of  s,  y,  and  x.  The  instrument  should  be  checked  up 
for  each  series  of  analyses  by  reading  the  standard  against  itself  and 
determining  the  potential  height  of  the  standard  solution  by  reading 
the  scale  on  the  left  side  when  the  solution  on  the  right  side  is  kept  at  a 
definite  height,  and  the  two  are  matched. 

I.  Acetone  Bodies. — Nephelometric  Methods  of  Marriott ^ — Prin- 
ciple.— Acetone  in  very  small  amounts  forms  a  cloudy  solution  with  the 
Scott-Wilson  reagent  which  may  be  read  nephelometrically.     By  this 

^  Kober:  Jour.  Ind.  and  Eng.  Chem.,  7,  843,  1915. 

^  The  instrument  is  manufactured  by  Lenz  and  Naumann  Pullman  Building,  17  Madison 
Ave.,  Xew  York  City. 

*  Kober:  /.  Am.  Chem.  Soc,  37,  2379,  1915;  Jour.  Biol.  Chem.,  13,  485,  1913. 

*  Marriott:  Jour.  Biol.  Chem.,  16,  289  and  293,  1913. 


BLOOD   ANALYSIS  295 

method  it  is  possible  to  make  a  complete  analysis  for  acetone  and 
diacetic  acid  and  hydroxybutyric  acid  in  from  2-5  c.c.  of  blood. 

Procedure.- — Two  to  5  c.c.  of  blood  drawn  from  a  superficial  arm  vein  by  means 
of  a  sterile  syringe  are  run  into  a  small  weighed  flask  containing  50  c.c.  of  0.5 
per  cent  potassium  oxalate  solution.  The  flask  is  reweighed.  The  diluted  blood 
is  run  into  100  c.c.  of  boiUng  water  acidified  with  i  c.c.  of  glacial  acetic  acid' 
contained  in  an  800  c.c.  Kjeldahl  distilling  flask  and  the  procedure  is  then  carried 
out  as  described  in  the  Marriott-Scott -Wilson  methods  for  (a.)  acetone  and  diac- 
etic acid  and  (b)  /3-hydroxybutyric  acid  as  outlined  on  page  284.  The  precipitate 
in  the  mercury  reagent  is  however  estimated  nephelemometrically.  In  this  case 
the  distillate  which  should  measure  75-100  c.c.  is  allowed  to  stand  half  an  hour, 
then  transferred  to  a  graduated  cyUnder  and  diluted  until  an  opalescence  that 
can  be  conveniently  read  is  obtained.  The  turbidity  occasioned  by  0.05  mg.  of 
acetone  diluted  to  100  c.c.  is  a  convenient  strength  for  this  purpose,  although 
considerably  larger  or  smaller  amounts  give  good  results.  With  heavy  opales- 
cence it  is  desirable  after  diluting  to  a  certain  volmne,  say  250  c.c.  to  remove 
an  aliquot  portion  with  a  pipette  and  dilute  this  appropriately.  A  solution  con- 
taining a  known  amount  of  acetone-  is  distilled  into  an  excess  of  reagent^  and 
this  distillate  which  is  to  be  used  as  the  standard  is  diluted  as  above.  Read  in  the 
nephelometer  against  this  standard,  taking  the  readings  as  quickly  as  possible 
after  filling  the  tubes  as  the  suspension  settles  slowly. 

If  the  unknown  suspension  is  diluted  so  as  to  be  not  more  than  20  per  cent 
different  from  the  standard  and  if  comparisons  are  made  in  considerable  depths 
of  solution  (50-60  mm.)  no  corrections  are  necessary.  If  the  two  agree  within 
10  per  cent  accurate  comparison  may  be  made  at  less  depths.  If  divergences  are 
greater  and  accurate  results  are  desired,  Kober's  correction  equation  must  be 
used  (see  discussion  of  nephelometer  p.  294). 

2.  Fat. — Nephelometric  Method  of  Bloor^ — Principle. — The  protein  is  precipi- 
tated with  alcohol  and  ether  and  the  fatty  acid  in  the  extract  determined  nephelo- 
metrically  after  saponification. 

Procedure. — Extraction. — About  2  c.c.  of  blood  are  drawn  from  the  vein  with  a 
graduated  syringe  and  run  at  once  with  stirring  into  a  weighed  graduated  flask 
containing  about  40  volumes  of  a  mixture  of  3  parts  alcohol  and  i  part  ether. 
After  again  weighing  to  find  the  weight  of  blood  added,  the  solution  is  raised  to 
boiling  in  a  water-bath,  cooled  under  the  tap,  made  to  volume  with  alcohol-ether 
mixture,  mixed  and  filtered.     The  filtrate  is  water  clear  and  almost  colorless. 

Determination. — From  5-20  c.c.  of  the  extract  (containing  about  2  mg.  of  fat) 
are  measured  with  a  pipette  into  a  small  beaker  and  saponified  b)'-  evaporating 
nearly  but  not  quite  to  dryness  with  2  c.c.  of  N/i  sodium  ethylate.  The  residue  is 
heated  just  to  boiling  after  the  addition  of  5  c.c.  of  alcohol-ether,  and  50  c.c.  of 
distilled  water  are  added. 

A  similar  solution  of  the  standard  is  prepared  by  adding  5  c.c.  of  the  standard 

^  Commercial  varieties  of  acetic  acid  frequently  contain  substances  which  behave  like 
acetone.     Blank  determinations  should  always  be  made  and  corrections  made  accordingly. 

2  A  convenient  stock  solution  contains  about  0.03  mg.  acetone  per  c.c.  The  strength 
of  such  a  solution  is  determined  by  titration  of  200  c.c.  bv  the  Messincer-Huppert  method 
(see  Chapter  XXVI). 

'  The  solution  cannot  be  added  directly  to  the  reagent  as  a  lower  result  is  obtained  than 
when  distilled. 

*Bloor:  Jour.  Biol.  CItcm.,  17,  377,  1914;  23.  31;,  IQ15. 


296  PHYSIOLOGICAL    CHEMISTRY 

fatty  acid  solution^  from  a  pipette  with  stirring  to  50  c.c.  of  distilled  water.  To 
the  standard  and  to  the  test  solutions  are  added  simultaneously  from  pipettes  and 
with  stirring  10  c.c.  portions  of  dilute  (1:3)  hydrochloric  acid  and  the  solutions 
allowed  to  stand  for  five  minutes,  after  which  they  are  transferred  to  the  comparison 
tubes  of  the  nephelometer  (see  Fig.  83,  p.  291).  Several  readings  should  be  taken 
and  averaged.  The  standard  tube  should  always  be  on  the  same  side.  See  dis- 
cussion of  nephelometer  (page  290)  for  details  as  to  reading.  The  results  repre- 
sent the  amount  of  total  fat  (fatty  acids  and  cholesterol)  in  the  blood,  expressed 
as  oleic  acid.  The  fat  of  the  corpuscles  is  not  completely  extracted,  and  it  should 
be  borne  in  mind  that  other  lipoids  as  cholesterol  are  included  in  the  results. 
Cholesterol  may  be  determined  separately  and  subtracted  from  the  result  for  total 
fat.  It  may  also  be  determined  in  a  part  of  the  blood  extract  as  prepared  above 
by  a  modified  Autenrieth-Funk  procedure. ^  Methods  have  also  been  devised  for 
the  determination  of  the  phosphatides  of  blood. ^ 

Instruments  Used  in  the  Examination  of  the  Blood 

Spectroscope.^ — Either  the  angular -vision  spectroscope  (Figs.  87 
and  88)  or  the  direct-vision  spectroscope  (Fig.  86)  may  be  used  in 
making  the  spectroscopic  examination  of  the  blood.  For  a  complete 
description  of  these  instruments  the  student  is  referred  to  any  standard 
text-book  of  physics. 


I'lG.  8().-  —  I)iRECT-\isi()N   Spectroscope. 

1.  Oxyhemoglobin. — Examine  dilute  (1:50)  defibrinated  blood  spectro- 
scopically.  Note  the  broad  absorption  band  between  D  and  E.  Continue  the 
dilution  until  this  single  broad  band  gives  place  to  two  narrow  bands,  the  one 
nearer  the  D  line  being  the  narrower.  These  are  the  typical  absorption  bands 
of  oxyhemoglobin  obtained  from  dilute  solutions  of  blood.  Now  dilute  the  blood 
very  freely  and  note  that  the  bands  gradually  become  more  narrow  and,  if  the 
dilution  is  sufficiently  great,  they  finally  entirely  disappear. 

2.  Hemoglobin  (so-called  Reduced  Hemoglobin). — To  blood  which  has  been 
diluted  sufficiently  to  show  well-defined  oxyhemoglobin  absorption  bands  add  a 
small  amount  of  Stokes'  reagent.^    The  blood  immediately  changes  in  color  from 

1  The  standard  solution  used  is  an  alcohol-ether  solution  of  pure  oleic  acid  of  which 
5  c.c.  contain  about  2  mg.  of  the  acid.     The  alcohol  and  ether  used  for  the  standard  are 
freshly  redistilled  absolute  alcohol  and  pure  dry  ether. 
^Bloor:  Jour.  Biol.  C/tem.,  23,  317,  1915. 
3  Greenwald:  Jour.  Biol.  Cliem.,  21,  29,  1915. 
Bloor:  Jour.  Biol.  Chem.,  22,  133,  1915,  23,  317,  1915. 
Kober  and  Egerer:  J.  Am.  Chem.  Soc,  37,  2373,  1915. 
Taylor  and  Miller:  Jour.  Biol.  Chem.,  18,  215,  1914. 
For  other  nephelometric  methods  see  Chapters  XVII  and  XXVI. 
■•  For  Absorption  Spectra  see  Plates  I  and  II. 

^  Stokes'  reagent  is  a  solution  containing  2  per  cent  ferrous  sulphate  and  3  per  cent  tar- 
taric acid.  When  needed  for  use  a  small  amount  should  be  placed  in  a  test-tube  and  ammo- 
nium hydroxide  added  until  the  precipitate  which  forms  on  the  first  addition  of  the  hydrox- 
ide has  entirely  dissolved.     This  produces  ammonium  ferrotartrate  which  is  a  reducing  agent. 


BLOOD    ANALYSIS 


297 


a  bright  red  to  violet-red.  The  oxyhemoglobin  has  been  reduced  through  the 
action  of  Stokes'  reagent  and  hemoglobin  (so-called  reduced  hemoglobin)  has 
been  formed.     This  has  been  brought  about  by  the  removal  of  some  of  the  loosely 


Fig.  87. — AxouLAR-visioN  Spectroscope  Arranged  for  .\bsorptiox  Analysis. 

combined  oxygen  from  the  oxyhemoglobin.  Examine  this  hemoglobin  spectro- 
scopically.  Note  that  in  place  of  the  two  absorption  bands  of  oxyhemoglobin  we 
now  have  a  single  broad  band  lying  almost  entirely  between  D  and  E.  This  is 
the  typical  spectrum  of  hemoglobin.     If  the  solution  showing  this  spectrum  be 


Fig.  88. — Diagram  of  Angular-vision  Spectroscope.     (Long.) 

The  white  light  F  enters  the  collimator  tube  through  a  narrow  slit  and  passes  to  the 
prism,  P,  which  has  the  power  of  refracting  and  dispersing  the  light.  The  raj-s  then  pass  to 
the  double  conve.x  lens  of  the  ocular  tube  and  arc  deflected  to  the  eyepiece  E.  The  dotted 
lines  show  the  magnified  virtual  image  which  is  formed.  The  third  tube  contains  a  scale 
whose  image  is  reflected  into  the  ocular  and  shown  with  the  spectrum.  Between  the  light 
F  and  the  collimator  slit  is  placed  a  cell  to  hold  the  solution  undergoing  examination. 

shaken  in  the  air  for  a  few  moments  it  will  again  assume  the  bright  red  color  of 
oxyhemoglobin  and  show  the  characteristic  spectrum  of  that  pigment. 

3.  Carbon  Monoxide  Hemoglobin.  The  preparation  of  this  pigment  may  be 
easily  accompUshed  by  passing  ordinary  illuminating  gas'  through  defibrinated 

^  The  so-called  water  gas  with  which  ordinary  illuminating  gas  is  diluted  contains  usu- 
ally as  much  as  20  per  cent  of  carbon  monoxide  (CO). 


298  PHYSIOLOGICAL   CHEMISTRY 

OX -blood.  Blood  thus  treated  assumes  a  brighter  tint  (carmine)  than  that  im- 
parted by  oxyhemoglobin.  In  very  dilute  solution  oxyhemoglobin  appears 
yellowish  red  whereas  carbon  monoxide  hemoglobin  under  the  same  conditions 
appears  bluish  red.  Examine  the  carbon  monoxide  hemoglobin  solution  spec- 
troscopically.  Observe  that  the  spectrum  of  this  body  resembles  the  spectnim 
of  oxyhemoglobin  in  showing  two  absorption  bands  between  D  and  E.  The 
bands  of  carbon  monoxide  hemoglobin,  however,  are  somewhat  nearer  the  violet 
end  of  the  spectrimi.  Add  some  Stokes'  reagent  to  the  solution  and  again  ex- 
amine spectroscopically.  Note  that  the  position  and  intensity  of  the  absorption 
bands  remain  unaltered. 

The  following  is  a  delicate  chemical  test^  for  the  detection  of  carbon  monoxide 
hemoglobin : 

Tannin  Test. — ^Divide  the  blood  to  be  tested  into  two  portions  and  dilute  each 
with  4  volumes  of  distilled  water.  Place  the  diluted  blood  mixtures  in  two  small 
flasks  or  large  test-tubes  and  add  20  drops  of  a  lo  per  cent  solution  of  potassiimi 
ferricyanide.2  Allow  both  solutions  to  stand  for  a  few  minutes,  then  stopper  the 
vessels  and  shake  one  vigorously  for  10-15  minutes,  occasionally  removing  the 
stopper  to  permit  air  to  enter  the  vessel.'  Add  5-10  drops  of  amLmonium  svdphide 
(yellow)  and  10  c.c.  of  a  10  per  cent  solution  of  tannin  to  each  flask.  The  contents 
of  the  shaken  flask  will  soon  exhibit  the  formation  of  a  dirty  oUve-green  precipitate, 
whereas  the  flask  which  was  not  shaken  and  which,  therefore,  still  contains  car- 
bon monoxide  hemoglobin,  will  exhibit  a  bright  red  precipitate,  characteristic  of 
carbon  monoxide  hemoglobin.  This  test  is  more  deUcate  than  the  spectroscopic 
test  and  serves  to  detect  the  presence  of  as  low  a  content  as  5  per  cent  of  carbon 
monoxide  hemoglobin. 

4.  Neutral  Methemoglobin. — Dilute  a  Uttle  defibrinated  blood  (i  :  10)  and 
add  a  few  drops  of  a  freshly  prepared  10  per  cent  solution  of  potassium  ferricya- 
nide.  Shake  this  mixture  and  observe  that  the  bright  red  color  of  the  blood  is 
displaced  by  a  brownish  red.  Now  dilute  a  Uttle  of  this  solution  and  examine  it 
spectroscopically.  Note  the  single,  very  dark  absorption  band  lying  to  the  left 
of  D,  and,  if  the  dilution  is  sufficiently  great,  also  observe  the  two  rather  faint 
bands  lying  between  D  and  E  in  somewhat  similar  positions  to  those  occupied  by 
the  absorption  bands  of  oxyhemoglobin.  Add  a  few  drops  of  Stokes'  reagent  to 
the  methemoglobin  solution  while  it  is  in  position  before  the  spectroscope  and  note 
the  immediate  appearance  of  the  oxyhemoglobin  spectrum  which  is  quickly  fol- 
lowed by  that  of  hemoglobiti. 

5.  Alkaline  Methemoglobin. — Render  a  neutral  solution  of  methemoglobin, 
such  as  that  used  in  the  last  experiment  (4),  slightly  alkaline  with  a  few  drops  of 
ammonia.  The  solution  becomes  redder  in  color,  due  to  the  formation  of  alkaline 
methemoglobin  and  shows  a  spectrum  different  from  that  of  the  neutral  body.  In 
this  case  we  have  a  band  on  either  side  of  D,  the  one  nearer  the  red  end  of  the 
spectrum  being  much  the  fainter.  A  third  band,  darker  than  either  of  those  men- 
tioned, lies  between  D  and  E  somewhat  nearer  E. 

6.  AlkaU  Hematin. — Observe  the  spectrum  of  the  alkali  hcmatin  prepared  in 
Experiment  18  on  page  266.     Also  make  a  spectroscopic  examination  of  a  freshly 

^  Sand  (Ugeskrifl  for  Laeger,  76,  1721,  1914;  Abst.  J.  A.  M.  A.,  Nov.  21,  1914)  proposes 
a  potassium  iodide  test  for  carbon  monoxide  hemoglobin  in  blood.  He  claims  0.125  per 
cent  may  be  detected  by  his  test. 

^This  transforms  the  oxyhemoglobin  into  methemoglobin. 

'  This  is  done  to  free  the  blood  from  carbon  monoxide  hemoglobin. 


BLOOD   ANALYSIS  299 

prepared  alkali  hematin.'     The  typical  spectrum  of  alkali  hematin  shows  a  single 
absorption  band  lying  across  D  and  mainly  toward  the  red  end  of  the  spectrum. 

7.  Reduced  Alkali  Hematin  or  Hemochromogen. — Dilute  the  alkali  hematin 
solution  used  in  the  last  experiment  (6)  to  such  an  extent  that  it  shows  no  absorption 
band.  Now  add  a  few  drops  of  Stokes'  reagent  or  ammonium  sulphide  and  note 
that  the  greenish-brown  color  of  the  alkali  hematin  solution  is  displaced  by  a 
bright  red  color.  This  is  due  to  the  formation  of  hemochromogen  or  reduced 
alkali  hematin.  Examine  this  solution  spectroscopically  and  observe  the  narrow, 
dark  absorption  band  lying  midway  between  D  and  E.  If  the  dilution  is  not 
too  great  a  faint  band  may  be  observed  in  the  green  extending  across  E  and  b. 

8.  Acid  Hematin. — To  some  defibrinated  blood  add  half  its  volume  of  glacial 
acid  and  an  equal  volume  of  ether.  Mix  thoroughly.  The  acidified  etheral 
solution  of  hematin  rises  to  the  top  and  may  be  poured  off  and  used  for  the  spectro- 
scopic examination.  If  desired  it  may  be  diluted  with  acidified  ether  in  the  ratio 
of  one  part  of  glacial  acetic  acid  to  two  parts  of  ether.  A  distinct  absorption  band 
will  be  noted  in  the  red  between  C  and  D  and  lying  somewhat  nearer  C  than  the 
band  in  the  methemoglobin  spectrum.  Between  D  and  F  may  be  seen  a  rather 
indistinct  broad  band.  Dilute  the  solution  untU  this  band  resolves  itself  into  two 
bands.  Of  these  the  more  prominent  is  a  broad,  dark  absorption  band  lying  in  the 
green  between  b  and  F.  The  second,  a  narrow  band  of  faint  outline,  lies  in  the 
light  green  to  the  red  side  of  E.  A  fourth  very  faint  band  may  be  observed  lying 
on  the  violet  side  of  D. 

9.  Acid  Hematoporphyrin. — To  5  c.c.  of  concentrated  sulphuric  acid  in  a  test- 
tube  add  2  drops  of  blood,  mixing  thoroughly  by  agitation  after  the  addition  of 
each  drop.  A  wine-red  solution  is  produced.  Examine  this  solution  spectroscopic- 
ally. Acid  hematoporphyrin  gives  a  spectrum  with  an  absorption  band  on  either 
side  of  D,  the  one  nearer  the  red  end  of  the  spectrum  being  the  narrower. 

10.  Alkaline  Hematoporphyrin. — Introduce  the  acid  hematoporphyrin  solution 
just  examined  into  an  excess  of  distilled  water.  Cool  the  solution  and  add  potas- 
sium hydroxide  slowly  until  the  reaction  is  but  slightly  acid.  A  colored  precipitate 
forms  which  includes  the  principal  portion  of  the  hematoporphyrin.  The  presence 
of  sodium  acetate  facilitates  the  formation  of  this  precipitate.  Filter  off  the 
precipitate  and  dissolve  it  in  a  small  amount  of  dilute  potassium  hydroxide.  Alka- 
line hematoporphyrin  prepared  in  this  way  forms  a  bright  red  solution  and  possesses 
four  absorption-bands.  The  first  is  a  very  faint,  narrow  band  in  the  red,  midway 
between  C  and  D ;  the  second  is  a  broader,  darker  band  lying  across  D,  principally  to 
the  violet  side.  The  third  absorption  band  lies  principally  between  D  and  E,  ex- 
tending for  a  short  distance  across  E  to  the  violet  side,  and  the  fourth  band  is 
broad  and  dark  and  lies  between  b  and  F.  The  first  band  mentioned  is  the  faintest 
of  the  four  and  is  the  first  to  disappear  when  the  solution  is  diluted. 

I.  Fleischl's  Hemometer  (Fig.  89,  p.  300). — This  is  an  instrument 
used  quite  extensively  clinically,  for  the  quantitative  determination  of 
hemoglobin.  The  instrument  consists  of  a  small  cylinder  which  is  pro- 
vided with  a  fixed  glass  bottom  and  a  movable  glass  cover,  and  which  is 

1  Alkali  hematin  may  be  prepareil  by  mixing  one  volume  of  a  concentrated  potassium 
hydroxide  or  sodium  hydro.xide  solution  and  two  volumes  of  dilute  (i :  5)  delibrinated  blood. 
This  mbcture  should  be  heated  gradually  almost  to  boiling,  then  cooled  and  shaken  for 
a  few  moments  in  the  air  before  examination. 


300 


PHYSIOLOGICAL    CHEMISTRY 


Fig. 


divided,  by  means  of  a  metal  septum,  into  two  compartments  of  equal 
capacity.  This  cylinder  is  supported  in  a  vertical  position  by  means  of 
a  mechanism  which  resembles  the  base  and  stage  of  an  ordinary  micro- 
scope. Underneath  the  stage  is  placed  a  colored  glass  wedge  (see 
Fig.  91),  so  arranged  as  to  run  immediately  beneath  the  glass 
bottom  of  one  of  the  compartments  of  the  cylinder  and  ground  in  such  a 
manner  that  each  part  of  the  wedge  corresponds  in  color  to  a  solution 

of  hemoglobin  of  some  definite  percent- 
age. The  glass  wedge  is  held  in  a  metal 
frame  and  may  be  moved  backward  or 
forward  by  means  of  a  rack  and  pinion 
arrangement.  A  scale  along  the  side  of 
this  frame  indicates  the  percentage  of  the 
normal  amount  of  hemoglobin  which  each 
particular  variation  in  the  depth  of  color 
of  the  ground  wedge  represents,  taking 
the  normal  hemoglobin  content  as  100.^ 
In  a  position  corresponding  to  the  posi- 

-Fleischl's  Hemometer.   tion  of  the  mirror  on  the  ordinary  micro- 

{Da  Costa.)  .  .  ^ 

scope  is  attached  a  light-colored  opaque 
plate  which  serves  to  reflect  the  light  upward  through  the  colored 
wedge  and  the  cylinder  to  the  eye  of  the  observer. 

In  making  a  determination  of  the  percentage  of  hemoglobin  by  this  instru- 
ment the  procedure  is  as  follows:  Fill  each  compartment  about  three-fourths 
full  of  distilled  water.  Puncture  the  finger-tip  or  lobe  of  the  ear  of  the  subject  by 
means  of  a  sterile  needle  or  scalpel  and,  as  soon  as  a  drop  of  blood  appears, 
place  one  end  of  the  capillary  pipette  (Fig.  90),  which  accompanies  the  instrument, 
against  the  drop  and  allow  it  to  fill  by  capillary  attraction.  To  prevent  the  blood 
from  adhering  to  the  exterior  of  the  tube,  and  so  render  the  determination  inac- 
curate, it  is  customary  to  apply  a  very  thin  coating  of  mutton  fat  to  the  outer  sur- 
face before  using  or  to  wrap  the  tube  in  a  piece  of  oily  chamois  when  not  in  use. 
As  soon  as  the  tube  has  been  accurately  filled  with  blood  it  should  be  dipped  into 
the  water  of  one  of  the  compartments  of  the  cylinder  and  all  traces  of  the  blood 
washed  out  with  water  by  means  of  a  small  dropper  which  accompanies  the 
instrument.  If  the  blood  is  not  well  distributed  throughout  the  compartment  and 
does  not  form  a  homogeneous  solution  the  contents  of  the  compartment  should  be 
mixed  thoroughly  by  means  of  the  metal  handle  of  the  capillary  measuring  pipette. 
When  this  has  been  done  each  compartment  should  be  completely  filled  with  dis- 
tilled water  and  the  glass  cover  adjusted,  care  being  taken  that  the  contents  of 
the  two  compartments  do  not  mix.  Now  adjust  the  cylinder  so  that  the  compart- 
ment containing  the  pure  distilled  water  is  immediately  above  the  colored  glass 
wedge.  By  means  of  the  rack  and  pinion  arrangement  manipulate  the  colored 
wedge  until  a  portion  of  it  is  found  which  corresponds  in  color  with  the  diluted 
blood.     When  this  agreement  in  color  has  been  secured  the  point  on  the  scale  cor- 

'  The  scale  of  the  ordinary  instrument  is  usually  too  high. 


BLOOD    ANALYSIS 


301 


Fig.  90. — Pipette 
OF       Fleischl's 

HEMOiCETER. 


responding  to  this  particular  color  should  be  read  and  the  actual  percentage  of 
hemoglobin  computed.  For  instance,  if  the  scale  reading  is  90  it  means  that  the 
blood  under  examination  contains  90  per  cent  of  the  normal  quantity  of  hemo- 
globin, i.e.,  90  per  cent  of  14  per  cent. 

2.  Fleischl-Miescher  Hemometer.— The  apparatus  of  Fleischl 
has  been  modihed  by  Miescher.  If  all  precautions  are  taken,  the 
margin  of  error  in  the  absolute  quantity  of  hemoglobin  determined  by 
this  instrument  does  not  exceed  0.15-0.22  per  cent  by  weight  of  the 
blood.  Detailed  directions  for  the  manipulation  of 
the  Fleischl-Miescher  hemometer  accompany  the  in- 
strument. In  brief  Miescher  modified  the  instru- 
ment as  follows:  (i)  The  scale  of  each  instrument 
is  supplied  with  a  caliber  table  of  absolute  hemo- 
globin values,  expressed  in  milligrams:  the  scale  of 
Fleischl's  hemometer  shows  the  percentage  of  hemo- 
globin in  relation  to  an  average  selected  somewhat 
arbitrarily.  Thus  many  errors  arising  from  the  irregular  coloring  of  the 
glass  wedge  of  the  older  apparatus  are  avoided  in  the  instrument  as 
modified.  (2)  Each  instrument  is  accompanied  by  a  measuring  pip- 
ette (melangeur)  which  allows  of  a  more  accurate  measurement  of  the 
blood  than  was  possible  with  the  capillary  tubes  of  the  older  appara- 
tus, (jj  With  the  aid  of  the  measuring  pipette  mentioned  above 
blood  of  varying  degrees  of  concentration  may  be  compared.  In  this 
way  the  indi\ddual  examinations  are  controlled  and  a  check  upon  the 

accuracy  of  the  graduation  in  the 


color  of  the  glass  wedge  is  also 
afforded.  This  wedge  is  much 
more  evenly  and  accurately  colored 
than  in  the  unmodified  apparatus 
of  Fleischl.  (4)  Before  reading 
the  percentage  as  indicated  by  the 
scale,  the  chamber  is  covered  with 
a  glass  and  a  diaphragm  which  sharply  define  the  field  on  all  sides 
without  the  formation  of  a  meniscus. 

The  measuring  pipette  is  constructed  essentially  the  same  as  the 
pipettes  which  accompany  the  Thoma-Zeiss  apparatus  (see  page  305). 
The  capillary  portion,  however,  is  graduated,  i,  2/3  and  1/2  which 
enables  the  observer  to  dilute  the  blood  sample  in  the  proportion  of 
I  :20o,  I  :3oo  or  i  :40o  as  he  may  desire.  If  there  is  difficulty  in 
drawing  in  the  blood  exactly  to  one  of  the  graduations  just  mentioned 
the  amount  of  blood  above  or  below  the  volume  indicated  by  the  gradu- 
ation may  be  determined  by  means  of  certain  delicate  cross-lines  which 


Fig.    91. — Colored     Glass     Wedge     of 
Fleischl's  Hemometer.     (Da  Costa.) 


302 


PHYSIOLOGICAL    CHEMISTRY 


are  placed  directly  above  and  below  the  graduation.  Each  cross-line 
corresponds  to  i/ioo  of  the  volume  of  the  capillary  tube  from  the  tip 
to  the  I  graduation. 

A  O.I  per  cent  solution  of  sodium  carbonate  is  used  to  dissolve  the 
stroma  of  the  erythrocytes  and  so  render  the  blood  solution  perfectly 
clear.  If  this  is  not  done  the  color  of  the  blood  solution  invariably  ap- 
pears darker  in  tone  than  that  of 
the  colored  glass  wedge.  A  freshly 
prepared  sodium  carbonate  solu- 
tion should  be  used  in  order  that 
the  clearness  of  the  solution  may 
not  be  marred  by  the  presence  of 
sodium  bicarbonate. 

3.  Dare's  Hemoglobinometer 
(Fig.  92). — This  instrument,  as 
the  name  signifies,  is  used  for  the 
determination  of  hemoglobin.  In 
using  either  Fleischl's  hemometer 
or  the  instrument  as  modified  by 
Miescher  the  blood  is  diluted  for 
examination,  whereas  with  the 
Dare  instrument  no  dilution  is  re- 
quired. This  probably  allows  of 
rather  more  accurate  determina- 
tions than  are  possible  with  the 
old  Fleischl  apparatus. 

The  instrument  consists  essen- 
tially of  the  following  parts:  (i) 
A  capillary  observation  cell,  (2)  a 
semicircular  colored  glass  wedge, 
(3)  a  milled  wheel  for  manipulat- 
ing the  wedge,  (4)  a  candle  used 
to  illuminate  portions  of  the  capillary  observation  cell  and  the  colored 
wedge,  (5)  a  small  telescope  used  in  the  examination  of  the  areas  illumi- 
nated by  the  candle  flame,  (6)  a  scale  graduated  in  percentages  of  the 
normal  amount  of  hemoglobin,  (7)  a  hard-rubber  case,  (8)  a  movable 
screen  attached  to  the  case. 

The  capillary  observation  cell  is  formed  of  two  small,  polished 
rectangular  plates  of  glass,  one  being  transparent  and  the  other  opaque. 
When  held  in  position  on  the  instrument,  by  means  of  a  small  metal 
bracket,  the  opaque  portion  of  the  cell  is  nearer  the  candle  and  thus 
serves  to  soften  the  glare  of  light  when  an  observation  is  being  made. 


Fig.    92. — Dare's  Hemoglobinometer. 
{,Da  Costa.) 

R,  Milled  wheel  acting  by  a  friction  bear- 
ing on  the  rim  of  the  color  disc;  S,  case  in- 
closing color  disc,  and  provided  with  a  stage 
to  which  the  blood  chamber  is  fitted;  T, 
movable  wing  which  is  swung  outward  dur- 
ing the  observation,  to  serve  as  a  screen  for 
the  observer's  eyes,  and  which  acts  as  a 
cover  to  inclose  the  color  disc  when  the  in- 
strument is  not  in  use;  U,  telescoping  cam- 
era tube,  in  position  for  examination;  V, 
aperture  admitting  light  for  illumination  of 
the  color  disc;  X,  capillary  blood  chamber 
adjusted  to  stage  of  instrument,  the  slip  of 
opaque  glass,  W,  being  nearest  to  the  source 
of  light;  Y,  detachable  candle-holder;  Z, 
rectangular  slot  through  which  the  hemo- 
globin scale  indicated  on  the  rim  of  the 
color  disc  is  read. 


BLOOD    ANALYSIS 


303 


The  transparent  portion  of  the  cell  is  directly  over  a  circular  opening 
in  the  case,  through  which  the  blood  specimen  is  viewed  by  means  of 
the  small  telescope. 

The  semicircular  colored  glass  wedge  is  so  ground  that  each  par- 
ticular shade  of  color  corresponds  to  that  possessed  by  fresh  blood  which 
contains  some  definite  percentage  of  hemo- 
globin. It  is  mounted  upon  a  disc  which 
may  be  manipulated  by  the  milled  wheel  in 
such  a  manner  as  to  bring  successive  por- 
tions of  the  wedge  in  position  to  be  viewed 
through  a  circular  opening  contiguous  to  the 
opening  through  which  the  blood  specimen  is 
viewed.  For  a  further  description  of  the  in- 
strument see  Figs.  92,  93,  and  94. 

In  using  the  Dare  hemoglobinometer  proceed  as 
follows :  Puncture  the  finger-tip  or  lobe  of  the  ear 
of  the  subject  by  means  of  a  needle  or  scalpel  and, 
after  a  drop  of  blood  of  good  proportions  has 
formed,  place  the  fiat  capillary  observation  cell  in 
contact  with  the  drop  and  aUow  it  to  fill  by  cap- 
illary attraction  (Fig.  94).     Replace  the  cell  in  its 

proper  place  on  the  instnunent.  When  in  position,  a  portion  of  this  cell  may  be 
observed  through  a  small  telescope  attached  to  the  apparatus.  It  is  viewed 
through  a  circular  opening  and  near  this  circle  is  a  second  one  through  which  a 
portion  of  a  semicircvilar  colored  glass  wedge  is  visible.  These  two  circles  are 
illuminated  simultaneously  by  means  of  the  flame  of  a  candle.     The  colored  glass 


Fig.  93. — Horizontal  Sec- 
tion OF  Dare's  Hemoglobin- 
ometer.    (Da  Cosia.) 


Fig.  04. — Method  of  Filling  the  Capillary  Observatiojsj  Cell  of  Dare's  Hemo- 
globinometer.    (Da  Costa.) 


may  be  rotated  by  means  of  a  milled  wheel  and  the  point  of  agreement  of  the 
color  of  the  adjoining  discs  may  be  determined  in  the  same  way  as  in  Fleischl's 
hemometer.  The  scale  reading  gives  the  percentage  of  the  normal  quantity 
of  hemoglobin  which  the  blood  sample  imder  examination  contains.  Compute 
the  actual  hemoglobin  content  in  the  same  manner  as  from  the  scale  reading  of 
the  Fleischl  hemometer  (see  page  299). 


304 


PHYSIOLOGICAL    CHEMISTRY 


4.  Tallquist's  Hemoglobin  Scale. — This  consists  essentially  of  a 
series  of  ten  colors  corresponding  to  stains  produced  by  blood  contain- 
ing varying  percentages  of  hemoglobin. 

In  using  this  scale  a  drop  of  blood  is  allowed  to  fall  on  a  small  section  of 
filter  paper  and  the  resulting  color  is  compared  with  the  ten  colors  of  the  scale. 
When  the  color  in  the  scale  is  found  which  corresponds  to  the  color  of  the  blood 
stain  the  accompanying  hemoglobin  value  is  read  off  directly. 

This  is  a  very  convenient  method  for  determining  hemoglobin  at  the 
bedside.  There  is  a  possibility  of  the  colors  being  inaccurately  printed, 
however,  and  even  if  originally  correct  in  tint,  under  the  continued 
influence  of  air  and  light  they  must  eventually  alter  somewhat. 

5.  Thoma-Zeiss  Hemoc3rtometer. — This  is  an  instrument  used  in 
"blood  counting,"  i.e.,  in  determining  the  number  of  erythrocytes  and 
leucocytes.  The  instrument  consists  of  a  microscopic  slide  constructed 
of  heavy  glass  and  provided  with  a  central  counting  cell  (see  Fig.  95, 


C' 


Fig.  95. — Thoma-Zeiss  Counting  Chamber.     {Da  Costa.) 

below).  This  cell,  with  the  cover  glass  in  position,  is  exactly  o.i  mm. 
deep.  The  floor  of  the  cell  is  divided  by  delicate  lines  into  squares  each 
of  which  is  1/400  sq.  mm.  in  area  (see  Fig.  97,  page  306).  The  volume 
of  blood,  therefore,  between  any  particular  square  and  the  cover  glass 
above  must  be  1/4000  cu.  mm.  Accompanying  each  instrument  are 
two  capillary  pipettes  (Fig.  96),  each  constructed  with  a  mixing  bulb 
in  its  upper  portion.  Each  bulb  is  further  provided  with  an  enclosed 
glass  bead  which  is  of  great  assistance  in  mixing  the  contents  of  the 
chamber.  The  stem  of  each  pipette  is  graduated  in  tenths  from  the 
tip  to  the  bulb.  The  final  graduation  at  the  upper  end  of  the  bulb  is 
1 01  on  the  pipette  used  in  mixing  the  blood  sample  in  which  the  ery- 
throcytes are  counted  (erythrocytometer,  see  Fig.  96),  and  11  on 
the  pipette  used  in  mixing  the  blood  sample  for  the  leucocyte  count 
(leucocytometer,  see  Fig.  96).  In  making  "blood  counts"  with  the 
hemocytometer  it  is  necessary  to  use  some  diluting  fluid.  Two  very 
satisfactory  forms  of  fluid   for  this  purpose  are  Toison's  and   Sher- 


BLOOD    ANALYSIS 


305 


rington's  solutions.^  When  either  of  these  solutions  is  used  as  the  dilut- 
ing fluid  it  is  possible  to  make  a  very  satisfactory  count  of  both  the 
erythrocytes  and  leucocytes  from  the  same  prepara- 
tion, since  the  leucocytes  are  stained  by  the  meth}l- 
violet  or  methylene-blue. 


In  counting  the  erythrocytes  by  means  of  the  hemo- 
cjrtometer,  proceed  as  follows  :  Thoroughly  cleanse  the  tip 
of  the  finger  or  lobe  of  the  ear  of  the  subject  by  the  use  of 
soap  and  water,  alcohol  and  ether  apphed  in  the  sequence 
just  given.  Puncture  the  skin  by  means  of  a  needle  or 
scalpel  and  allow  the  blood  drop  to  form  without  pressure. 
Place  the  tip  of  the  pipette  in  contact  with  the  blood  drop, 
being  careful  to  avoid  touching  the  skin,  and  draw  blood  into 
the  pipette  up  to  the  point  marked  0.5  or  i  according  to 
the  desired  dilution.  Rapidly  wipe  the  tip  of  the  pipette 
and  immediately  fill  it  to  the  point  marked  loi  with  Toison's 
or  Sherrington's  solution.  Now  thoroughly  mix  the  blood 
and  diluting  fluid  within  the  mixing  chamber  by  tapping 
the  pipette  gently  against  the  finger,  or  by  shaking  it  while 
held  securely  with  the  thumb  at  one  end  and  the  middle 
finger  at  the  other.  After  the  two  fluids  have  been 
thoroughly  mixed  the  diluting  fluid  contained  in  the  capil- 
lary-tube below  the  bulb  should  be  discarded  in  order  to  in- 
sure the  collection  of  a  drop  of  the  thoroughly  mixed  blood 
and  diluting  solution  for  examination.  Transfer  a  drop  from 
the  pipette  to  the  ruled  floor  of  the  counting  chamber  and, 
after  placing  the  cover-glass  firmly  in  position,-  allow  an  in- 
terval of  a  few  minutes  to  elapse  for  the  corpuscles  to  settle 
before  making  the  count.  Now  place  the  shde  under  the 
microscope  and  count  the  number  of  erythrocytes  in  a  num- 
ber of  squares,  counting  the  corpuscles  which  are  in  con- 
tact with  the  upper  and  the  right-hand  boundaries  of  the 


21 


w 


Fig.  96. — THOiL\- 
Zeiss  Capillary 
Pipettes. 

A,  Erythrocytometer; 
B,  Leucocytometer. 


Sherrington's   solution  has   the   following 
formula: 

Methylene-blue o.  i  gram. 

Sodium  chloride 1.2  gram. 

Neutral  potassium  o.xalate 1.2  gram. 

Distilled  water 300.0  grams. 


^  Toison's  solution  has   the  following 
formula: 

Methyl-violet 0.025  gram. 

Sodium  chloride i  gram. 

Sodium  sulphate 8  grams. 

Glycerol 30  grams. 

Distilled  water 160  grams. 

^  If  the  cover-glass  is  in  accurate  apposition  to  the  counting  cell  Newton's  rings  may  be 
plainly  observed.  Eustis  {Jour.  Ant.  Med.  Ass'n,  61,  1984,  1913)  suggests  the  following 
technic:  "After  the  usual  shaking  of  the  pipette,  and  expulsion  of  a  few  drops  of  the 
suspension,  a  good  sized  drop  is  placed  on  the  counting  chamber,  no  particular  atten- 
tion being  paid  to  its  size.  The  cover-glass,  which  has  been  previously  cleaned,  is 
then  rapidly  grasped  between  the  thumb  and  index-finger  of  the  right  hand,  while  the 
slide  is  steadied  on  the  table  with  the  left  hand.  While  firm  pressure  is  exerted  on  the 
cover-glass  it  is  rapidly  slid  across  the  counting  chamber,  through  the  drop  of  suspen- 
sion on  it.  The  cover-glass  will  cut  through  the  drop  at  exactly  o.i  mm.  The  excess 
from  the  drop  will  rise  on  top  of  the  cover-glass  and  jump  across  the  moat.  Newton's 
rings  will  be  obtained  in  each  instance.  The  drop  on  top  of  the  edge  of  the  cover- 
glass  is  wiped  or  soaked  up  with  the  point  of  a  towel  or  blotting-paper  and  the  preparation 
is  completed." 


3o6 


PHYSIOLOGICAL    CHEMISTRY 


square  as  belonging  to  that  square.  Take  the  squares  in  some  definite  se- 
quence in  order  that  the  recounting  of  the  same  corpuscles  may  be  avoided. 
A  satisfactory  procedure  is  to  begin  in  the  upper  right-hand  comer  and  proceed 
from  left  to  right  counting  the  cells  in  each  individual  square.  Take  the  next 
lower  row  of  squares  and  count  from  left  to  right  and  so  on  (see  Fig.  loi,  page 
311).  Of  course,  all  things  being  equal,  the  greater  the  number  of  squares  ex- 
amined the  more  accurate  the  count.  It  is  considered  essential  under  all  cir- 
cimistances,  where  an  accurate  count  is  desired,  that  the  counting  chamber 
shall  be  filled,  at  least  twice,  and  the  individual  counts  made  in  each  instance, 
as  indicated  above,  before  the  data  are  deemed  satisfactory.  Under  no  condi- 
tions should  less  than  200  squares  be  examined. 


Fig.  97. — Ordinary  Ruling  of  Thoma-Zeiss  Counting  Chamber.     {Da  Costa.) 

To  calculate  the  number  of  erythrocytes  per  cubic  millimeter  of 
undiluted  blood  proceed  as  follows:  Determine  the  number  of  cor- 
puscles in  any  given  number  of  squares  and  divide  this  total  by  the 
number  of  squares,  thus  obtaining  the  average  number  of  erythrocytes 
per  square.  Multiply  this  average  by  4000  to  obtain  the  number  of 
erythrocytes  per  cubic  millimeter  of  diluted  blood,  and  multiply  this 
product  by  100  or  200,  according  to  the  dilution,  to  obtain  the  number  of 
erythrocytes  per  cubic  millimeter  of  undiluted  blood.     Thus: 

per 


Average  number  of  erythrocytes  X4000X  2oo(or  loo' 
per  square  ^  ^ 


_    Number   of   erythrocytes 
~       cubic  millimeter. 


Great  care  should  be  taken  to  see  that  the  capillary  pipette  is  prop- 
erly cleaned.  After  using,  it  should  be  immediately  rinsed  out  with  the 
diluting  fluid,  then  with  water,  alcohol,  and  ether  in  the  sequence  given. 
Finally  dry  air  should  be  drawn  through  the  capillary  and  a  horsehair 
inserted  to  prevent  the  entrance  of  dust  particles. 

In  counting  leucocytes  by  means  of  the  hemocytometer  proceed  as  follows : 
As  mentioned  above,  if  the  diluting  fluid  is  either  Toison's  or  Sherrington's 


BLOOD   ANALYSIS 


307 


solution  the  leucocytes  may  be  counted  in  the  same  specimen  of  blood  in  which 
the  erythrocytes  are  counted.  When  this  is  done  it  is  customary  to  use  a  sUde 
provided  with  Zappert's  modified  niling  (Fig.  gSj.  This  method  is  rather  more 
accurate  than  the  older  one  of  counting  the  leucocytes  in  a  separate  specimen 
of  blood.  Furthermore,  it  is  obviously  preferable  to  count  both  the  eryth- 
rocytes and  the  leucocytes  from  the  same  blood  sample.  To  insure  accuracy 
the  nvunber  of  leucocytes  within  the  whole  ruled  region  should  be  determined  in 
dupUcate  blood  samples.  This  includes  the  examination  of  an  area  eighteen 
times  as  great  as  the  old  style  Thoma-Zeiss  central  ruUng.  This  region  then 
would  correspond  to  3600  of  the  small  squares  and,  if  duphcate  examinations  were 
»  made,  the  total  bumber  of  small  squares  examined  would  aggregate  7200. 

The  calculation  would  be  as  follows : 

Number  of  leucocvtes  in  7200   ,,       , ,  .  Number    of    leucocytes  per    cubic 

'  X  200X4000  — 7200=  •„•      . 

squares  ^        ^-^t  1  millimeter.  1 


Fig.  98. — Zappert's  Modified  Ruling  OF  Thoma-Zeiss  Counting  Chamber.   iDa  Cosla.) 

If  a  Zappert  slide  is  not  available,  a  good  plan  to  follow  is  to  place  a 
diaphragm  in  the  tube  of  the  ocular  of  the  microscope  consisting  of  a 
circle  of  black  cardboard  or  metaP  having  a  square  hole  in  the  center  of 
such  a  size  as  to  allow  of  the  examination  of  exactly  100  squares  or  one- 
fourth  of  a  square  millimeter  at  one  time.  With  this  arrangement  any 
portion  of  the  specimen  may  be  examined  and  counted  whether  within 
or  without  the  ruled  area.  In  counting  by  means  of  this  dcNice  it  is,  of 
course,  helpful  if  the  microscope  is  provided  with  a  mechanical  stage, 
but  even  without  this  arrangement,  if  the  observer  is  careful  to  see  that 
the  leucocytes  at  the  extreme  boundary  of  one  field  move  to  the  opposite 
boundary  when  the  position  of  the  slide  is  changed,  the  device  may  be 
very  satisfactorily  employed.     The  leucocytes  should  be  counted  in  36 


'  Ehrlich's  mechanical  eyepiece  with   iris  diaphragm  is  al-- 
purpose. 


!li-;fart(>r\-  for  tills 


3o8  PHYSIOLOGICAL   CHEMISTRY 

of  the  diaphragm-fields  in  duplicate  specimens  and  the  calculation  made 
•  in  the  same  manner  as  explained  above. 

If  the  leucocytes  are  counted  in  a  separate  specimen  of  blood  ordina- 
rily the  diluting  fluid  is  0.3-0.5  per  cent  acetic  acid,  a  fluid  in  which  the 
leucocytes  alone  remain  visible.  Under  these  conditions  the  dilution  is 
customarily  made  in  the  pipette  having  1 1  as  the  final  graduation.  The 
capillary  portion  is  of  larger  caliber  and  so  requires  a  greater  amount  of 
blood  to  fill  it  to  the  0.5  or  i  mark  than  is  required  in  the  use  of  the  other 
form  of  pipette.  In  counting  the  leucocytes  according  to  this  method  it 
is  customary  to  draw  blood  into  the  pipette  up  to  the  i  mark  and  im- 
mediately fill  the  remaining  portion  of  the  apparatus  to  the  1 1  gradua- 
tion with  the  0.3-0.5  per  cent  acetic  acid.  It  then  remains  to  count  the 
number  of  leucocytes  in  the  whole  central  ruled  portion  of  400  squares. 
This  should  be  done  in  duplicate  samples  and  the  calculation  made  as 
follows : 

Number    of    leucocytes    in   800  ^  ^       .  o  Number    of    leucocytes   per    cubic 

X  4.000  X I o -^  80c  =  .,,.  -  ^ 

squares  ^j,^'^'^^^.^     ow^  millimeter. 

6.  Biirker's  Hemocytometer.^ — This  is  an  improved  apparatus^  for  the  more 
accurate  counting  of  erythrocytes  than  is  possible  by  the  Thoma-Zeiss  apparatus. 
The  principles  involved  are  somewhat  different  from  those  in  force  with  the  latter 
apparatus.  For  example,  the  blood  is  diluted  in  a  separate  vessel,  not  in  the  pipette 
with  which  the  sample  is  drawn,  and  furthermore  the  cover-g'ass  is  applied  to  the 
counting  chamber  and  damped  in  place  before  the  diluted  blood  is  applied  to  the 
ruled  area.  Hayem's  solution*  is  used  as  the  diluting  fluid.  Toison's  solution  is 
not  satisfactory  for  use  with  the  Biirker  counting  chamber  as  its  viscosity  is  too 
great.  The  corpuscles  settle  rapidly  in  Hayem's  fluid  as  the  specific  gravity  of  the 
fluid  is  1015  whereas  that  of  the  erythrocj'tes  is  1090. 

The  pipette  for  measuring  the  quantity  of  blood  (Fig.  99,  upper  pipette)  has  a 
point  which  is  not  ground  dull  but  is  polished.  This  allows  of  better  judgment  in 
deciding  whether  the  column  of  blood  extends  to  the  very  tip.  The  volume  of 
the  pipette  between  tip  and  mark  is  25  cu.  mm.  The  mark  extends  all  the  way 
around  the  tube  so  that  errors  of  parallax  may  be  avoided. 

The  pipette  for  measuring  the  diluting  fluid  (Fig.  99,  middle  pipette)  also  has 
a  polished  point  and  circular  mark  and  delivers  4975  cu.  mm.  This  volume  of 
diluting  fluid  with  25  cu.  mm.  of  blood  gives  a  dilution  of  i  :  200.  Both  pipettes 
are  provided  with  a  piece  of  rubber  tubing  and  mouthpiece. 

For  transferring  the  diluted  blood  from  the  diluting  flask  to  the  chamber  a 
plain  pipette  provided  with  a  rubber  cap  is  used  (Fig.  99,  lower  pipette).  It  is 
filled  by  pressing  the  cap  slowly  with  the  index-finger,  inserting  the  tip  into  the 
liquid  and  then  releasing  the  pressure. 

*  Biirker:  Pfliiger's  Archiv.,  142,  337,  191 1;  Miincli.  med.  Woch.,  59,  pp.  14  and  89,  1912. 

^  Manufactured  by  C.  Zeiss,  Jena. 

'  Hayem's  solution  has  the  following  formula: 

Mercuric  chloride o.  25  gram. 

Sodium  chloride ^ o.    5  gram. 

Sodium  sulphate 2  .   5  grams. 

Distilled  water 100 .   o  grams. 


BLOOD    ANALYSIS 


309 


The  diluting  is  done  in  a  small  round-boUomed  flask  as  shown  in  Fig.  99. 
Several  of  these  flasks  should  be  kept  on  hand  in  a  wooden  rack  which  will  hold 
them  in  an  upright  position.  Each  flask  is  provided  with  a  paraflined,y)r  smooth 
cork  stopper. 

In  the  older  counting  chambers  the  floor  of  the  chamber  is  circular  and  the 
counting  is  done  in  the  center  of  this  space.  The  corpuscles  are  therefore  counted 
in  the  center  of  a  capillary,  circular  film  where  on  account  of  surface  tension  their 
number  is  slightly  greater  than  elsewhere.  This  source  of  error  is  avoided  in  the 
new  counting  chamber  (Fig.  102)  in  which  the  floor  is  represented  by  the  upper 
surface  of  a  piece  of  glass  25  mm.  long  and  5  mm.  wide  which  is  rounded  off  at  both 
ends  and  divided  into  two  portions  by  a  groove  1.5  mm.  wide  through  the  center. 


Fig.  99. — BiJRKER's  Pipettes,  Mixing  Flasks  and  Counting  Chamber. 

At  each  side  of  this  floor  piece,  separated  from  it  by  a  groove  is  a  glass  plate 
(7.5  mm.  X  21  mm.)  of  such  height  that  the  space  between  the  floor  of  the  cell 
and  a  cover-glass  placed  across  the  plates  is  o.ioo  mm.  A  cover-glass  23  mm. 
long  and  21  mm.  wide  with  rounded  polished  edges  is  used  so  that  the  rounded 
ends  of  the  floor  piece  project  beyond  it.  The  chamber  is  provided  with  clamps 
to  press  the  cover-glass  firmly  upon  both  plates  (Fig.  99). 

The  ruling  on  each  portion  of  the  floor  piece  is  that  shown  in  Fig.  100,  which 
will  be  explained  below\ 

Measuring  the  Diluting  Fluid. — Four  thousand  nine  hundred  and  seventy-five 
cu.  mm.  of  diluting  fluid  {Ilayenis)  are  measured  out  into  the  diluting  flask.  To 
do  this  the  pipette  is  filled  by  suction  to  slightly  above  the  mark  and  the  rubber 
tube  is  carefully  clamped  off.  Then  with  a  soft  piece  of  linen  the  tip  is  wiped  dr>'. 
The  meniscus  is  then  accurately  adjusted  to  the  mark  by  lightly  touching  the  point 
of  the  pipette  to  the  cleaned  tip  of  the  finger.     The  pipette  is  then  inserted  into 


3IO 


PHYSIOLOGICAL   CHEMISTRY 


the  diluting  flask  and  with  the  tip  nearly  touching  the  bottom  of  the  flask  the  fluid 
is  allowed  to  run  out.  The  time  of  the  flow  should  be  about  forty  seconds  and  is 
controlled  by  placing  the  tip  of  the  index-finger  loosely  upon  the  mouthpiece. 
The  pipette  is  emptied  completely  by  alternately  blowing  through  it  and  touching 
it  to  the  wall  of  the  flask  slightly  above  the  level  of  the  liquid.  The  drops  clinging 
to  the  wall  are  united  with  the  bulk  of  the  liquid  by  a  suitable  motion  of  the  flask. 
The  flask  is  then  stoppered,  care  being  taken  from  now  on  that  none  of  the  liquid 
ever  touches  the  neck  of  the  flask  or  the  stopper. 

Taking  the  Blood  Sample. — Usually  the  best  time  to  draw  the  blood  is  before 
breakfast.  For  a  single  determination  the  author  prefers  to  draw  it  from  the  tip 
of  the  fourth  finger  of  the  left  hand.  For  repeated  determinations  it  is  well  to 
change  off  between  third,  fourth  and  fifth  fingers  of  left  hand.  The  temperature 
of  the  room  should  not  be  below  i7°C.  to  prevent  an  undue  contraction  of  the 
cutaneous  vessels.  The  instrument  used  to  puncture  the  finger  should  have  a 
chisel-shaped  point  which  is  preferable  to  the  ordinary  lancet-shaped  point.     The 


Fig.  ioo. — Ruling  of  Burker  Counting  Chamber. 


first  drop  of  blood  is  wiped  off.  Into  the  second  one  the  tip  of  the  pipette  is  in- 
serted and  blood  is  drawn  in  until  the  meniscus  is  even  with  or  a  little  beyond  the 
mark.  The  tip  is  then  wiped  off  without  touching  the  capillary  opening  and  the 
observer  assures  himself  that  the  column  of  blood  extends  to  the  very  end  of  the 
capillary.     The  meniscus  is  then  accurately  adjusted  to  the  mark. 

Mixing  of  the  Blood  and  Diluting  Fluids. — The  tip  of  the  pipette  is  now  dipped 
into  the  diluting  fluid  which  has  been  measured  into  the  flask  and  the  blood  is 
slowly  blown  out.  The  blood  having  a  much  higher  specific  gravity  than  the 
Hayem's  fluid  sinks  to  the  bottom.  The  pipette  is  then  filled  with  the  pure  super- 
natant diluting  fluid  and  emptied  again,  care  being  taken  to  avoid  air  bubbles. 
This  is  repeated  until  the  blood  is  removed  as  completely  as  possible.  To  mix  the 
blood  and  diluting  fluid  the  flask  is  rotated  for  two  minutes  in  spiral  curves  of 
continually  decreasing  radius.  The  motion  should  be  alternately  clockwise  and 
counterclockwise.  After  complete  mixing  the  pipette  is  rinsed  out  several  times 
with  the  diluted  blood. 


BLOOD    ANALYSIS 


311 


Transferral  of  the  Diluted  Blood  to  the  Chamber. — The  counting  chamber  which 
has  been  cleaned  with  distilled  water  and  alcohol-ether  and  then  wiped  dry  with 
a  soft  cloth  as  free  from  lint  as  possible  is  placed  upon  a  black  surface  and  carefully 
brushed  with  a  camel's  hair  brush.  The  cover-glass  is  now  placed  over  the  chamber 
by  sliding  it  over  the  two  glass  plates  with  both  thumbs  while  the  index-fingers  are 
pressing  it  down.  By  means  of  the  clamps  it  is  held  in  place  firmly  so  that  New- 
ton's rings  (if  possible  of  the  first  order:  brown  and  black)  may  be  seen  over  the 
entire  area  of  the  plates.  The  chamber  is  placed  upon  the  stage  of  the  microscope 
and  is  brought  into  a  horizontal  position. 

Before  transferring  the  diluted  blood  to  the  chamber  the  flask  must  be  shaken 
for  two  minutes  as  described  before.  The  liquid  shows  a  cloudy  appearance  and 
must  be  allowed  to  stand  until  the  turbidity  has  become  uniform. 

One  of  the  plain  pipettes  described  above  is  now  inserted  into  the  diluted  blood 


I 

5 

T 

9 

13 

-1 

1 

— » 

-- 

^ 

-5 

5 

-^ 

- 

— * 

-9 

9 

-^ 

- 

— > 

-13 

13 

1 

> 

T 

£ 

» 

1: 

J 

Fig.  ioi. — Schema. 


while  slight  pressure  is  being  exerted  on  the  rubber  cap.  The  pressure  is  released 
slowly  and  the  liquid  rises  into  the  pipette.  The  point  of  the  pipette  is  now  im- 
mediately placed  upon  one  of  the  projecting  ends  of  the  floor  plate  and  verj-  slight 
pressure  is  exerted  on  the  rubber  cap  until  the  liquid  coming  from  the  pipette  just 
reaches  the  cover-glass  when  the  pressure  is  released.  An  instantaneous  filling 
of  the  capillary  space  results.  The  pipette  should  be  emptied  immediately,  rinsed 
with  distilled  water  and  placed  in  an  upright  position  in  a  beaker  of  water.  The 
other  portion  of  the  counting  chamber  is  now  filled  in  the  same  way  with  a  second 
pipette  and  about  one  minute  is  allowed  for  the  settb'ng  of  the  corpuscles.  During 
this  time  the  pipettes  may  be  washed  with  distilled  water  and  ether-alcohol  and 
dried  by  suction.  Occasionally,  the  pipettes  should  be  cleaned  with  a  horse  hair 
and  with  concentrated  H2SO4  containing  a  little  KjCraO?, 

To  see  whether  the  distribution  of  the  corpuscles  has  been  uniform  the  chamber 
is  illuminated  with  a  wide-open  diaphragm  and  viewed  at  an  angle.     If  the  opacity 


312 


PHYSIOLOGICAL   CHEMISTRY 


is  not  uniform  in  either  of  the  portions  of  the  chamber,  that  one  should  not  be  used 
for  counting.  If  the  counting  must  be  interrupted  or  requires  a  long  time  a  moist 
chamber^  should  be  used  to  prevent  evaporation  of  the  diluting  fluid.  The  diluted 
blood  may  be  retained  in  the  mixing  flasks  and  duplicate  countings  obtained  after 
the  lapse  of  twenty-four  hours  or  more  according  to  Biirker. 

Counting  and  Calculation. — A  mechanical  stage  movable  in  two  directions  is 
indispensable.  With  a  magnification  of  320  diameters  the  counting  is  begun  in 
the  left  upper  corner  of  the  ruling.  Proceed  from  left  to  right  along  one  row  then 
move  from  right  to  left  along  the  next  lower  row,  and  so  on.  Only  the  small 
squares  are  used  for  counting  (see  Fig.  100)  and  the  figures  are  recorded  in  the 
schema'^  (see  Fig.  loi)  in  which  the  squares  crossed  by  horizontal  or  vertical  lines 
correspond  to  the  small  squares  used  for  counting.  Usually  80  squares  are  counted 
and  by  recording  the  figures  in  the  schema  the  count  may  be  verified  and  an  idea  of 


Tiefe 

O.lOOmm. 

C.Zeiss 
Jerva 

0 

O.Olmm. 

Fig.  102. — BtJRKER  Counting  Chamber. 


the  uniformity  of  the  distribution  may  be  formed.  Half  of  the  counted  squares 
should  be  in  the  one,  half  in  the  other  portion  of  the  counting  chamber.  For 
more  accurate  measurements  more  squares  may  be  counted. 

The  observer  will  do  well  not  to  attempt  counting  each  individual  corpuscle 
in  a  square.  After  some  practice  each  typical  group  of  corpuscles  will  immediately 
suggest  a  number.  A  very  common  form  of  grouping  is  one  corpuscle  surrounded 
by  four  others.  This  should  immediately  suggest  the  number  five.  In  this  way 
the  counting  will  become  more  rapid  and  also  more  reliable. 

The  calculation  is  very  simple.  The  number  of  corpuscles  in  80  squares 
divided  by  100  will  give  the  numBer  of  millions  per  cubic  millimeter.  If,  for 
example,  536  corpuscles  have  been  counted  in  80  squares  then  with  a  dilution 
of     I  :  200  the   number  of  corpuscles   per  cubic  millimeter  is  5,360,000.     Thus, 

536 

Q—X 4000X200=  5,360,000  erythrocytes  per  cubic   miUimeter.     More  than  two 

oO 

decimal  places  are  without  significance. 

1  Biirker:  Pfliiger's  Archiv,  118,  465,  1907. 

2  The  firm  of  H.  Laupp  in  Tubingen  has  put  this  schema  on  the  market  (in  packs  of  100). 


CHAPTER  XVII 
MILK 

Milk  is  the  most  satisfactory  individual  food  material  elaborated  by 
nature.  It  contains  the  three  nutrients,  protein,  fat,  and  carbohydrate 
and  inorganic  salts  in  such  proportion  as  to  render  it  a  very  acceptable 
dietary  constituent.  It  is  a  specific  product  of  the  secretory  activity  of 
the  mammary  gland.  It  contains,  as  the  principal  solids,  olein, 
pahnitin,  stearin,  hutyrin,  casein,  lad-alhumin,  laclo-glohulin,  lactose, 
phosphates  of  calcium,  potassium  and  magnesium,  citrates  of  sodium 
and  potassium  and  chloride  of  calcium.  The  calcium  phosphate  of 
milk  is  the  neutral  calcium  phosphate,  CaHP04.^  ]Milk  also  contains 
some  iron  but  not  enough  for  the  needs  of  the  body  if  milk  is  the  only 
source  of  the  iron.  It  also  contains  at  least  traces  of  lecithin,  cholesterol, 
urea,  creatine,  creatinine,  and  the  tri-glycerides  of  caproic,  lauric,  and 
myristic  acids.  According  to  Osborne  and  Wakeman-  milk  contains 
two  phosphatides,  one  being  probably  stearyl-oleyl-lecithin. 

Recent  investigations  indicate  the  presence  in  milk  of  some  sub- 
stance or  substances  of  unknown  character  which  are  of  great  nutritional 
importance.  The  presence  of  a  grouih-promoting  substance  (\'itamine) 
i7i  butter  fat  has  been  demonstrated  by  McCollom  and  Davis  and  by 
Osborne  and  Mendel.^ 

By  passing  milk  through  a  special  form  of  earthenware  filter  Van 
Slyke  and  Bosworth^  have  obtained  a  separation  of  the  constituents  in 
milk  which  are  in  true  solution  from  those  insoluble  in  water  or 
in  suspension.  The  soluble  constituents  and  the  water  constitute 
the  milk  serum.  They  suggest  the  following  classification  of  milk 
constituents: 

CONSTITUENTS  OF  MILK 


In  true  solution  in  milk  Partly  in  solution  and  Entirely    in    suspension 

serum.  partly  in  suspension  or  or    colloidal   solution. 

colloidal  solution. 

1.  Lactose.  i.  Albumin.  i.  Fat. 

2.  Citric  acid.  2.  Inorganic  phosphate.  2.  Casein. 

3.  Potassium.  3.  Calcium. 

4.  Sodium.  5.  Magnesium. 

5.  Chlorine. 

'Van  Slyke  and  Bosworth:  Jour.  Biol.  Chcm.,  20,  135,  1915. 
^Osborne  and  Wakeman:  Jour.  Biol.  Client.,  21,  539,  1915. 

*  McCollom  and  Davis;  Jour.  Biol.  Chem.,  15,  167,  1913;  Osborne  and  Mendel:  ibid, 
15,  311,  1913;  24,  37,  1916  (previous  references  cited  in  this  article). 

*  Van  Slyke  and  Bosworth:  Jour.  Biol.  Chcm.,  20,  135,  1915. 

3^3 


314  PHYSIOLOGICAL   CHEMISTRY 

Fresh  milk,  both  human  and  cow^s,  is  amphoteric  in  reaction  to 
litmus  and  acid  to  phenolphthalein.  The  acidity  is  believed  to  be  due 
in  part  at  least  to  soluble  acid  phosphates}  Upon  standing  for  a 
sufficiently  long  time,  unsterilized  milk  sours,  i.e.,  it  becomes  strongly 
acid  in  reaction  to  litmus  due  to  the  production  of  the  optically  in- 
active fermentation  lactic  acid, 

H    OH 
H— C— C— COOH, 

I  ! 

H    H 

from  the  lactose  contained  in  it.  This  is  brought  about  through 
bacterial  activity.  The  white  color  is  imparted  to  the  milk  partly 
through  the  fine  emulsion  of  the  fat  and  partly  through  the  medium  of 
the  caseinogen  in  solution.  The  specific  gravity  of  milk  varies  some- 
what, the  average  being  about  1.030.  Its  freezing-point  is  about 
-o.56°C. 

This  lactic  acid  fermentation  may  be  brought  about  by  Bad.  lactis 
and  other  microorganisms.  Certain  putrefactive  bacteria  in  the 
human  intestines  may  also  cause  lactic  acid  fermentation.  The  chem- 
ical changes  in  lactic  acid  fermentation  may  be  indicated  thus: 

C12H22O11-I-H2O — >C6Hi206-l-C6Hi206 

Lactose.  Galactose  Glucose. 

C6H12O6— ^sCsHeOs 

Galactose         Lactic  Acid, 
or  Glucose. 

Fresh  milk  does  not  coagulate  on  being  boiled  but  a  film  consisting 
of  a  combination  of  casein  and  calcium  salts  forms  on  the  surface. 
If  the  film  be  removed,  thus  allowing  a  fresh  surface  to  come  into 
contact  with  the  air,  a  new  film  will  form  indefinitely  upon  the  applica- 
tion of  heat.  Surface  evaporation  and  the  presence  of  fat  facilitate 
the  formation  of  the  film,  but  are  not  essential  (Rettger^) .  As  Jamison 
and  Hertz^  have  shown,  a  similar  film  will  form  on  heating  any  protein 
solution  containing  fat  or  paraffin.  If  the  milk  is  of  a  pronounced 
acid  reaction,  through  the  inception  of  lactic  acid  fermentation,  or  from 
any  other  cause,  no  film  will  form  when  heat  is  applied,  but  instead  a 
true  coagulation  will  occur.  When  milk  is  boiled  certain  changes  occur 
in  its  odor  and  taste.  These  changes,  according  to  Rettger,^  are  due 
to  a  partial  decomposition  of  the  milk  proteins  and  are  accompanied 
by  the  liberation  of  a  volatile  sulphide,  probably  hydrogen  sulphide. 

^  Rettger:  American  Journal  of  Physiology,  7,  325,  1902. 
^Jamison  and  Hertz:  Journal  of  Physiology,  27,  26,  1902. 
•Rettger:  American  Journal  of  Physiology,  6,  450,  1902. 


MILK 


3^5 


The  milk-curdling  enzymes  of  the  gastric  and  the  pancreatic  juice 
have  the  power  of  splitting  the  casein  of  the  milk,  through  a  process  of 
hydrolytic  cleavage,  into  soluble  paracasein  and  a  peptone-like  body. 
This  soluble  paracasein  then  forms  a  combination  with  the  soluble 
calcium  salts  of  the  milk  and  an  insoluble  curd  of  paracasein  results. 
The  clear  fluid  surrounding  the  curd  is  known  as  whey.  This  action 
of  rennin  may  be  represented  by  the  following  scheme: 

Casein  (+  rennin  > 


Peptone-like 
body 


Soluble  paracasein 

I 

-f-  Ca  salts 

I 
Paracasein 

(insoluble  curd) 


Fig.  103. — Normal  Milk  and  Colostrum. 
a,  Normal  milk;  b,  Colostrum. 

There  is  still  considerable  confusion  of  terms  when  different  authori- 
ties discuss  milk  proteins  and  the  action  of  milk  curdling  enzymes  upon 
them.  The  English  scientists^  quite  uniformly  call  the  principal  pro- 
tein of  milk  caseinogen  whereas  the  insoluble  curd  formed  by  rennin  is 
termed  casein.  On  the  other  hand,  the  Germans  and  many^^mericans 
give  the  name  casein  to  the  milk  protein  and  paracasein  to  the  product 
of  the  action  of  rennin  upon  this  protein.  The  confusion  of  terms 
may  be  represented  thus: 

English  German. 

Caseinogen.  =  Casein. 

Casein.  =  Paracasein. 

^Halliburton:  Journal  of  Physiology,  ir,  44S,  iqoo. 


3i6 


PHYSIOLOGICAL    CHEMISTRY 


The  most  important  difference  between  human  milk  and  cow's 
milk  is  in  the  protein  content,  although  there  are  also  differences  in  the 
carbohydrate  and  ash  and  likewise  striking  biological  differences  diffi- 
cult to  define  chemically.  It  has  been  shown  that  the  casein  of  human 
milk  differs  from  the  casein  of  cow's  milk  in  being  more  difficult  to 
precipitate  by  acid  or  coagulate  by  gastric  rennin.  The  casein  curd 
(paracasein)  also  forms  in  much  looser  and  more  flocculent  manner 
than  that  from  cow's  milk  and  is  for  this  reason  much  more  easily 
digested  than  the  latter.  Both  human  and  cow's  milk  contain  im- 
portant non-nitrogenous  substances  of  an  unknown  character.  Human 
milk  contains  the  greater  quantity  of  these  substances.^ 

The  relative  composition  of  human  and  cow's  milk  is  shown  in  the 
following  table  which  embraces  data  reported  by  Meigs  and  Marsh. 

COMPOSITION  OF  MILK  (PER  CENT  OF  WHOLE  MILK)  NORMAL  VARIATIONS 
FROM  BEGINNING  OF  SECOND  MONTH  OF  LACTATION 


Constituent 

Cow 

Human 

Water  (Avg.). 

87.0 

87-5 

Solids  (Avg.) . 

13.0 

12.5 

Protein 

4-2-5 

I -5-0- 7 

Fat 

2-4 

2-4 

Sugar 

3-5-5 

6-7-5 

Ash 

0 . 6-0 . 7 

0.2-0.3 

The  above  data  indicate  that  human  milk  contains  less  protein, 
more  sugar  and  much  less  ash  than  cow^s  milk.  The  percentage 
composition  of  human  milk  at  different  periods  is  represented  in  the 
following  table." 

PERCENTAGE  COMPOSITION  OF  HUMAN  MILK  BY  PERIODS 


Period 

Fat 

Sugar 

Protein 

Casein 

Albumin     Ash 

Total 
solids 

Colostrum  (1-12  days) 

. . .  .  2.8^ 

f.  en 

9.     2C 

0.31 

13-4 

Transition  (12-30  days) 

••■•4-37J  7-74  ;   1-56 

0. 24 

I^.A 

Mature  (1-9  raos.)  

...  .13.26 

7-50  j  i-iS 

0.43 

0.72 

0.21 

U-2 

Late  (10-20  mos.) 

....3.16 

1 

7.47 

1.07 

0.32 

0.75 

0.20      12.2 

^  Meigs  and  Marsh:  Jour.  Biol.  Chem.,  16,  147,  1913. 

'Holt,  Courtney  and  Fales:  Am.  Jour.  Dis.  Children,  10,  229,  1915. 


MILK 


317 


The  composition  of  the  ash  of  milk  is  shown  in  the  following  table 
reported  by  Holt,  Courtney  and  Fales.^ 

PERCENTAGE  COMPOSITION  OF  THE  ASH  OF  MILK 


CaO 

1 

MgO 

P2O5 

NaiO 

1  K2O      CI 

Human  milk 

233 

3-7 

16.6 

7.2 

28.3    16.5 

Cow's  milk 

23.5 

2.8 

26.5 

7.2 

24-9    13-6 

It  will  be  observed  that  the  composition  of  the  ash  of  the  two  varieties 
of  milk  is  about  the  same  except  for  phosphorus.  The  higher  phos- 
phorus content  in  the  case  of  cow's  milk  is  due  principally  to  the  fact 
that  the  milk  contains  a  higher  percentage  of  casein  ox  phospJio protein. 
It  should  be  borne  in  mind  that  cow's  milk  contains  on  the  average 
over  three  times  as  much  ash  as  human  milk.  Therefore  unless  cow's 
milk  has  been  diluted  with  more  than  twice  its  volume,  there  is  still 
present  as  high  a  concentration  of  the  inorganic  constituents  as  are 
present  in  normal  human  milk.  Hence  there  is  no  necessity  for  the 
addition  of  any  of  these  constituents  in  infant  feeding. 

Interesting  data  relative  to  the  composition  of  milk  from  various 
sources  may  be  gathered  from  the  following  table  which  was  compiled 
mainly  from  the  results  of  investigations  by  Proscher-  and  by  Abder- 
halden^  in  Bunge's  laboratory.  It  will  be  noted  that  the  composition 
of  the  milk  varies  directly  with  the  length  of  time  needed  for  the  young 
of  the  particular  species  to  double  in  weight. 


Species 


Period  in  which 
weight  of   the 

newborn  is 
doubled  (days) 


100  Parts  of  milk  contain 


Proteins      Salts    Calcium 


Phosphoric 
acid 


Man. . . 
Horse . . 
Cow. . . 
Goat.  . 
Sheep. . 
Pig.... 
Cat.... 
Dog. . . 
Rabbit. 


180 
60 

47 
22 

15 

14 

9- 

9 

6 


0.  2 

0  033 

0.4 

0. 124 

0.7 

0. 160 

0.8 

0.197 

0.8 

0.245 

0.8 

0.24Q 

1  .  0 

1-3 

0.455 

2-5 

0.891 

0.047 
0.I3I 

0.197 
0.284 
0.293 

o .  30S 


0.508 
0.997 


The  secretion  of  the  mammary  glands  of  the  newborn  of  both  sexes 
is  called  "witches'  milk."     The  name  is  centuries  old  and  evidently 

'  Holt,  Courtney  and  Fales:  Am.  Jour.  Dis.  Children,  10,  229,  1915. 

-  Proscher:  Zcit.f.  physiol.  Chemie,  24,  285,  1898. 

^  .\bderhalden:  Ibid.,  26,  487,  1899;  and  27,  pp.  40S  and  457,  1899. 


3i8 


PHYSIOLOGICAL   CHEMISTRY 


refers  to  the  mystery  of  the  useless  secretion.  Basch^  has  recently  sug- 
gested that  this  secretion  of  "witches  milk"  is  brought  about  by  the 
passage  of  hormones  (see  Chapter  on  Pancreatic  Digestion)  from  the 
blood  of  the  mother  to  the  fetus. 

Lactose,  the  principal  carbohydrate  constituent  of  milk,  is  an  impor- 
tant member  of  the  disaccharide  group.  It  occurs  only  in  milk,  except 
as  it  is  found  in  the  urine  of  women  during  pregnancy,  during  the  nurs- 
ing period,  and  soon  after  weaning;  it  also  occurs  in  the  urine  of  normal 
persons  after  the  ingestion  of  a  very  large  amount  of  lactose  in  the  food. 
It  is  not  derived  directly  from  the  blood,  but  is  a  specific  product  of  the 
cellular  activity  of  the  mammary  gland.  It  has  strong  reducing  power, 
is  dextro-rotatory  and  forms  an  osazone  with  phenylhydrazine.  Lac- 
tose is  not  fermentable  by  pure  yeast.     For  changes  which  lactose 


Fig.  104. — Lactose. 

undergoes  in  lactic  acid  fermentation  see  page  314.  The  crystalline 
form  of  lactose  is  shown  in  Fig.  104. 

Casein,  the  principal  protein  constituent  of  milk,  belongs  to  the 
group  of  phosphoproteins  and  contains  0.7  per  cent  of  phosphorus.^ 
It  has  acidic  properties  and  combines  with  bases  to  produce  salts.^ 
It  is  probably  present  in  milk  in  the  form  of  neutral  calcium  caseinate 
(Casein  Ca4).^  It  is  not  coagulable  upon  boiling  and  is  precipitated 
from  its  neutral  solution  by  certain  metallic  salts  as  well  as  upon  satu- 
ration with  sodium  chloride  or  magnesium  sulphate.  Its  acid  solu- 
tion is  precipitated  by  an  excess  of  mineral  acid. 

Lactalbumin  and  lacto-globulin,  the  protein  constituents  of  milk, 

^  Basch:  Milnch.  med.  Woch.,  58,  2266,  1911. 

^  Bosvvorth  and  Van  Slyke:  Jour.  Biol.  Chem.,  19,  67,  1914. 

^  Van  Slyke  and  Bosworth:  Jour.  Biol.  Chem.,  14,  207-227,  1914. 

*  Van  Slyke  and  Bosworth:  Jour.  Biol.  Chem.,  20,  135,  191 5. 


MILK  319 

next  in  importance  to  casein,  closely  Resemble  serum  albumin  and 
serum  globulin  in  their  general  properties. 

Butter  (milk  fat)  consists  in  large  part  of  olein  and  palmitin. 
Stearin,  butyrin,  caproin  and  traces  of  other  fats  are  also  present. 
When  butter  becomes  rancid  through  the  cleavage  of  certain  of  its 
constituent  fats  by  bacteria  the  odors  of  caproic  and  butyric  acids  are 
in  evidence. 

The  pigment  of  the  fat  of  cow's  milk  is  made  up  of  carotin  and 
xanthophylls.  The  principal  pigment  is  carotin,  an  unsaturated 
hydrocarbon  pigment  which  is  widely  distributed  in  plants.^  The 
pigment  of  the  fat  of  hufnan  milk  is  made  up  of  carotin  and  xantho- 
phylls in  about  equal  proportions.  Carotin  is  also  probably  the 
pigment  of  human  fat.  The  pigment  of  body  fat,  blood  serum,  corpus 
luteum  and  skin  secretions  of  the  cow  is  principally  carotin. 

Colostrum  is  the  liame  given  to  the  product  of  the  mammary  gland 
secreted  for  a  short  time  before  parturition  and  during  the  early  period 
of  lactation  (see  Fig.  103,  page  315).  It  is  yellowish  in  color,  contains 
more  solid  matter  than  ordinary  milk,  and  has  a  higher  specific  gravity 
(i. 040-1. 080).  The  most  striking  difference  between  colostrum  and 
ordinary  milk  is  the  high  percentage  of  lactalbumin  and  lacto-globulin 
in  the  former.  This  abnormality  in  the  protein  content  is  responsible 
for  the  coagulation  of  colostrum  upon  boiling. 

Such  enzymes  as  lipase,  amylase,  galactase,  catalase,  oxidases, 
peroxidases,  and  reductases  have  been  identified  in  milk,  but  not  all  of 
them  in  milk  of  the  same  species  of  animal. 

Among  the  principal  preservatives  used  in  connection  with  milk  are 
formaldehyde,  hydrogen  peroxide,  boric  acid,  borates,  salicylic  acid, 
and  salicylates.     The  use  of  milk  preservatives  is  illegal  in  most  states. 

Experiments  on  Milk 

1.  Reaction. — Test  the  reaction  of  fresh  cow's  milk  to  htmus,  phenolphthalein 
and  Congo  red. 

2.  Biuret  Test. — Make  the  biuret  test  according  to  directions  given  on 
page  98. 

3.  Microscopical  Examination. — Examine  fresh  whole  milk,  skimmed  or 
centrifugated  milk,  and  colostnmi  under  the  microscope.  Compare  the  micro- 
scopical appearance  with  Fig.  103,  page  315. 

4.  Specific  Gravity. — Determine  the  specific  gravity  of  both  whole  and 
skinmied  milk  (see  page  324).  Which  possesses  the  higher  specific  gravity? 
Explain  why  this  is  so. 

5.  Film  Formation. — Place  lo  c.c.  of  milk  in  a  small  beaker  and  boil  a  few 
minutes.     Note  the  formation  of  a  film.     Remove  the  film  and  heat  again.     Does 

'Palmer  and  Eckles:  Jour.  Biol.  Client.,  17,  191,  1914. 


320  PHYSIOLOGICAL    CHEMISTRY 

the  film  now  form?  Of  what  substance  is  this  fihn  composed?  The  biuret  test 
was  positive ;  why  do  we  not  get  a  coagulation  here  when  we  heat  to  boiling? 

6.  Coagulation  Test. — Place  about  5  c.c.  of  milk  in  a  test-tube,  acidify  sUghtly 
with  dilute  acetic  acid  and  heat  to  boiling.     Do  you  get  any  coagulation?     Why? 

7.  Action  of  Hot  Alkali. — To  a  Uttle  milk  in  a  test-tube  add  a  few  drops  of 
potassium  hydroxide  and  heat.  A  yellow  color  develops  and  gradually  deepens 
into  a  brown.  To  what  is  the  formation  of  this  color  due?  (See  Moore's  Test, 
Chapter  II.) 

8.  Test  for  Chlorides. — To  about  5  c.c.  of  milk  in  a  test-tube  add  a  few  drops 
of  very  dilute  nitric  acid  to  form  a  precipitate.  Filter  off  this  precipitate  and  test 
the  filtrate  for  chlorides.     Does  milk  contain  any  chlorides? 

9.  Guaiac  Test. — To  about  5  c.c.  of  water  in  a  test-tube  add  3  drops  of 
milk  and  enough  alcoholic  solution  of  guaiac  (strength  about  i  :  60)^  to  cause 
turbidity.  Thoroughly  mix  the  fluids  by  shaking  and  observe  any  change  which 
may  gradually  take  place  in  the  color  of  the  mixture.  If  no  blue  color  appears 
in  a  short  time,  heat  the  tube  gently  below  6o°C.  and  observe  whether  the 
color  reaction  is  hastened.  In  case  a  blue  color  does  not  appear  in  the  course 
of  a  few  minutes,  add  hydrogen  peroxide  or  old  turpentine,  drop  by  drop,  until 
the  color  is  observed. 

Fresh  milk  will  frequently  give  this  blue  color  v^hen  treated  w^ith  an 
alcoholic  solution  of  guaiac  without  the  addition  of  hydrogen  peroxide 
or  old  turpentine.  Those  milks  which  respond  positively,  fail  to  do  so 
after  boiling  15-20  seconds.  What  substances  beside  milk  respond  to 
this  test?     See  discussion  on  page  258. 

10.  Differentiation  of  Human  and  Cow's  Milk  (Modification  of  Bauer's  Test)  2. 
— Introduce  2  c.c.  of  fresh  himian  milk  into  a  50  c.c.  test-tube  and  2  c.c.  of  fresh 
cow's  milk  into  another  similar  tube.  Add  to  the  contents  of  each  tube  i  drop  of 
a  0.25  per  cent  aqueous  solution  of  nile-blue  sulphate  (Griibler).  Shake  the 
tubes  gently  and  permit  them  to  stand  undisturbed  for  10-30  minutes.  The 
milk  assimies  a  bluish  cast  in  each  case.  At  the  end  of  the  lo-minute  interval 
add  10  c.c.  of  ether  to  the  contents  of  each  tube  and  shake  very  thoroughly  for  one 
minute.  The  ether  extracts  the  pigment  from  the  human  milk,  leaving  the  milk 
white.  In  the  case  of  cow's  milk  the  ether  does  not  extract  the  dye  and  the  milk 
remains  bluish  in  color. 

11.  Tests  to  Differentiate  between  Raw  Milk  and  Heated  Milk. — 

(a)  Trikresol  Peroxidase  Reaction  (Kastle). — The  peroxidase  reaction  of 
milk  is  founded  upon  the  fact  that  small  ainounts  of  raw  milk  will  in- 
duce the  oxidation  of  various  leuco  compounds  by  hydrogen  peroxide. 
This  reaction  has  been  used  in  a  practical  way  as  the  most  convenient 
means  of  differentiating  between  raw  milk  and  heated  milk.  Many 
substances  have  been  employed  for  this  purpose,  e.g.,  guaiac,  para- 
phenylenedi amine,  ortol,  amidol,  etc.     Kastle  has  found  that  a  dilute 

'  Buckmaster  advises  the  use  of  an  alcoholic  solution  of  guaiaconic  acid  instead  of  an 
alcoholic  solution  of  guaiac  resin.     Guaiaconic  acid  is  a  constituent  of  guaiac  resin. 
^  Bauer:  Monatssch.  J.  Kindertieil,,  11,  474,  191 2-13. 


MILK  321 

solution  of  "trikresol"^  acts  as  a  sensitizing  agent  in  the  peroxidase 
reaction  and  offers  the  following  test  which  is  based  upon  this  fact. 

Procedure. — To  2-5  c.c.  of  raw  milk  in  a  test-tube  add  0.1-0.3  c.c.  of  M/io 
hydrogen  peroxide  and  i  c.c.  of  a  i  per  cent  solution  of  "trikresol."  A  slight 
though  unmistakable  yellow  color  will  be  observed  to  develop  throughout  the 
solution.  Repeat  the  test  using  milk  which  has  been  boiled  or  heated  to 
8o°C.  for  10-20  minutes  and  cooled,  and  note  that  no  yellow  color  is  produced. 

The  color  reaction  in  the  case  of  the  raw  milk  probably  results  from 
the  oxidation  of  the  cresols  by  the  hydrogen  peroxide.  The  first 
product  of  this  oxidation^  then  oxidizes  the  leuco  compound,  when  such 
is  present,  and  causes  the  color  observed. 

(b)  Benzidine  Peroxidase  Reaction  {Wilkinson  and  Peters).^ — To  10  c.c.  of  the 
milk  to  be  tested  add  2  c.c.  of  a  4  per  cent  alcoholic  solution  of  benzidine,  suffi- 
cient acetic  acid  to  coagulate  the  milk  (usually  2-3  drops)  and  finally  2  c.c.  of  a 
3  per  cent  solution  of  hydrogen  peroxide.  Raw  milk  yields  an  immediate  blue 
color.  In  adding  the  peroxide  it  is  best  to  permit  it  to  flow  slowly  down  the 
wall  of  the  vessel  containing  the  mixture  instead  of  allowing  it  to  mix  with  the 
milk.     Milk  which  has  been  heated  to  78°C.  or  above  remains  unchanged. 

12.  Satxiration  with  Magnesium  Sulphate. — Place  about  5  c.c.  of  milk  in  a 
test-tube  and  saturate  with  solid  magnesium  sulphate.     What  is  this  precipitate? 

13.  Influence  of  Gastric  Rennin  on  Milk. — Prepare  a  series  of  five  tubes  as 
follows : 

(a)  5  c.c.  of  fresh  milk  -f  0.2  per  cent  HCl  (add  drop  by  drop  xmtil  a  precipitate 
forms). 

(b)  5  c.c.  of  fresh  milk  +  5  drops  of  rennin  solution.* 

(c)  5  c.c.  of  fresh  milk  +  10  drops  of  0.5  per  cent  Na^iCOj. 

(d)  5  c.c.  of  fresh  milk  +  10  drops  of  ammonimn  oxalate. 

(e)  5  c.c.  of  fresh  milk  +  5  drops  of  02  per  cent  HCl. 

Now  to  each  of  the  tubes  (c),  (d)  and  (e)  add  5  drops  of  rennin  solution. 
Place  the  whole  series  of  five  tubes  at  40°C.  and  after  10-15  minutes  note  what 
is  occurring  in  the  different  tubes.     Give  a  reason  for  each  particular  result. 

14.  Preparation  of  Casein. — Fill  a  large  beaker  one-third  full  of  skimmed 
(or  centrifugated)  milk  and  dilute  it  with  an  equal  volume  of  water.  Add  dilute 
hydrochloric  acid  until  a  flocculent  precipitate  forms.  Stir  after  each  acidifica- 
tion and  do  not  add  an  excess  of  the  acid  as  the  precipitate  would  dissolve. 
Allow  the  precipitate  to  settle,  decant  the  supernatant  fluid,  and  reserve  it  for 
use  in  later  (15-18)  experiments.  Filter  off  the  precipitate  of  casein  and  re- 
move the  excess  of  moisture  by  pressing  it  between  filter  papers.  Transfer  the 
casein  to  a  small  beaker,  add  enough  95  per  cent  alcohol  to  cover  it  and  stir  for 
a  few  moments.  Filter,  and  press  the  precipitate  between  filter  papers  to  re- 
move the  alcohol.  Transfer  the  casein  again  to  a  small  dry  beaker,  cover  the 
precipitate  with  ether  and  heat  on  a  water-bath  for  ten  minutes,  stirring  con- 

^  "Trikresol"  is  the  trade  name  of  an  antiseptic  which  contains  the  three  cresols  in  ap- 
proximately equal  proportions. 

'^  Probably  some  organic  pero.xide  or  quinoid  compound. 

^  Wilkinson  and  Peters:  Z.  Nahr-Genussm.,  16,  No.  3,  p.  172. 

*  Any  commercial  rennin  or  rennet  preparation  or  an  extract  of  the  gastric  mucosa 
of  the  pig  may  be  employed. 


322 


PHYSIOLOGICAL    CHEMISTRY 


tinuously.  Filter  (reserve  the  filtrate  i,  and  press  the  precipitate  as  dry  as  possible 
between  filter  papers.  Open  the  papers  and  allow  the  ether  to  evaporate  spon- 
taneously. Grind  the  precipitate  to  a  powder  in  a  mortar.  Upon  the  casein 
prepared  in  this  way  make  the  following  tests : 

(a)  Solubility. — ^Try  the  solubihty  in  water,  sodiimi  chloride,  dilute  acid  and 

alkali. 

(b)  Millon's  Reaction. — Make  the  test  according  to  the  directions  given  on 

page  97. 

(c)  Biuret  Test. — Make  the  test  according  to  directions  given  on  page  98. 

(d)  GlyoxyUc  Acid  Reaction  (HopMns-Colej. — Make  the  test  according  to 
the  directions  given  on  page  98. 

(e)  Unoxidized  Sulphur. — Test  for  unoxidized  sulphur  according  to  the  di- 
rections given  on  page  108.  The  sulphur  content  of  casein  is  rather  low,  e.g., 
about  0.7  per  cent. 

(f )  Fusion  Test  for  Phosphorus.— Test  for  phosphorus  by  fusion  according  to 
directions  given  on  page  129.     Casein  contains  0.7  per  cent  of  phosphorus. 

15.  Coagulable  Proteins  of  Milk. — Place  the  filtrate  from  the  original  casein 
precipitate  in  a  casserole  and  heat,  on  a  wire  gauze,  over  a  free  flame.  As  the 
solution  concentrates,  a  coagultmi  consisting  of  lactalbumin  and  lactoglobulin 
will  form.  Continue  to  concentrate  the  solution  until  the  volume  is  about  one- 
half  that  of  the  original  solution.  Filter  off  the  coagulable  proteins  (reserve  the 
filtrate)  and  test  them  as  follows  : 

(a)  Millon's  Reaction. — Make  the  test  according  to  the  directions  given  on 

page  97. 

(b)  Biuret  Test. — Make  the  test  according  to  the  directions  given  on  page  98. 

(c)  GlyoxyUc  Acid  Reaction  fHopkins-Cole). — Make  the  test  according  to 
the  directions  given  on  page  98. 

16.  Detection  of  Calcivun  Phosphate. — ^Evaporate  the  filtrate  from  the 
coagulable  proteins,  on  a  water-bath,  until  crystals  begin  to  form.     It  may  be 

necessary  to  concentrate  to  15  c.c.  before  any  crys- 
^    ^'^^^       tallization  will  be  observed.      Cool  the  solution,  filter 
off  the  crystals  (reserve  the  filtrate),  and  test  them 
as  follows: 

(a)  Microscopical     Examination. — Examine    the 
crystals   and   compare   them  with  those  in  Fig.  105. 

(b)  Dissolve    the   crystals   in   nitric   acid.     Test 
part  of  the  acid  solution  for  phosphates.     Render 

Fig.  105.     Calcium       ^he  remainder  of  the  solution  slightly  alkaline  with 
ammonia,  then  acidify  with  acetic  acid  and  add  am- 
moniimi  oxalate.     Examine  the  crystals  xmder  the  microscope  and  compare 
them  with  those  in  Fig.  134,  page  459. 

17.  Detection  of  Lactose. — Concentrate  the  filtrate  from  the  calcium  phos- 
phate until  it  is  of  a  syrup-like  consistency.  Allow  it  to  stand  over  night  and 
observe  the  formation  of  crystals  of  lactose.     Make  the  following  experiments. 

(a)  Microscopical  Examination. — Examine  the  crystals  and  compare  them 
with  those  in  Fig.  104,  page  318. 

(b)  Fehling's  Test.— Try  Fehling's  test  upon  the  mother  liquor. 

(c)  Phenylhydrazine  Test. — Apply  the  phenylhydrazine  test  to  some  of  the 
mother  liquor  according  to  the  directions  given  on  page  22. 


.MILK 


323 


18.  Milk  Fat. — (a)  Evaporate  the  ether  filtrate  from  the  casein  (Experiment 
13)  and  observe  the  fatty  residue.  The  milk  fat  was  carried  down  with  the 
precipitate  of  casein  and  was  removed  when  the  latter  was  treated  with  ether. 
If  centrifugated  milk  was  used  in  the  preparation  of  the  caseinogen  the  amount 
of  fat  in  the  ether  filtrate  may  be  very  small.  To  secure  a  larger  yield  of  fat 
proceed  according  to  directions  given  under  (b)  below. 

(b)  To  25  c.c.  of  whole  milk  in  an  evaporating  dish  add  a  Uttle  sand  or  filter 
paper  and  evaporate  the  fluid  to  dryness  on  a  water-bath.  Grind  or  break  up 
the  residue  after  cooUng  and  extract  with  ether  in  a  flask.  Filter  and  remove 
the  ether  from  the  filtrate  by  evaporation.  How  can  you  identify  fats  in  the 
ethereal  residue? 

19.  Saponification  of  Butter. — Dissolve  a  small  amount  of  butter  in  alcohol 
made  strongly  alkaUne  with  potassivmi  hydroxide.  Place  the  alcohoUc-potash 
solution  in  a  casserole,  add  about  100  c.c.  of  water  and  boil  for  10-15  minutes  or 
until  the  odor  of  alcohol  cannot  be  detected.  Place  the  casserole  in  a  hood  and 
neutraUze  the  solution  with  sulphuric  acid.  Note  the  odor  of  volatile  fatty  acids, 
particularly  butyric  acid.  Under  certain  conditions  the  odor  of  ethyl  butjrrate 
may  also  be  detected. 

20.  Detection  of  Preservatives. —  (a)  Formaldehyde. — In  these 
tests  tivo  controls  should  be  run,  one  with  pure  milk  and  one  with 
milk  to  which  a  very  small  amount  of  formaldehyde  has  been  added. 

I.  Leach's  Hydrochloric  Acid  Test. — Mix  10  c.c.  of  milk  and  10  c.c.  of  con- 
centrated hydrochloric  acid  containing  about  0.002  gram  of  ferric  chloride  in  a 
small  procelain  evaporating  dish  or  casserole  and  gradually  raise  the  temperature 
of  the  mixture,  on  a  water-bath,  nearly  to  the  boiling-point,  with  occasional 
stirring.  If  formaldehyde  is  present  a  violet  color  is  produced,  while  a  brown 
color  develops  in  the  absence  of  formaldehyde.  In  case  of  doubt  the  mixture, 
after  having  been  heated  nearly  to  the  boiling-point  for  about  one  minute, 
should  be  diluted  with  50-75  c.c.  of  water,  and  the  color  of  the  diluted  fluid 
carefully  noted,  since  the  violet  color  if  present  will  quickly  disappear.  For- 
maldehyde may  be  detected  by  this  test  when  present  in  the  proportion  i :  250,000. 

II.  Gallic  Acid  Test. — Acidify  30  c.c.  of  milk  with  2  c.c.  of  normal  sulphuric 
acid  and  distil.  Add  0.2-0.3  c.c.  of  a  saturated  alcoholic  solution  of  gallic  acid 
to  the  first  5  c.c.  of  the  distillate,  then  incline  the  test-tube  and  slowly  introduce 
3-5  c.c.  of  concentrated  sulphuric  acid,  allowing  it  to  run  slowly  down  the  side- 
of  the  tube.  A  green  ring,  which  finally  changes  to  blue,  is  formed  at  the  junc- 
ture of  the  fluids.  This  is  claimed,  by  Sherman,  to  be  twice  as  delicate  as  either 
the  sulphuric  acid  or  the  hydrochloric  acid  test  for  formaldehyde. 

{b)  Salicylic  Acid  and  Salicylates. — Remont's  Method.' — Acidify  20  c.c.  of  milk 
with  sulphuric  acid,  shake  well  to  break  up  the  curd,  add  25  c.c.  of  ether,  mix  thor- 
oughly, and  allow  the  mixture  to  stand.  By  means  of  a  pipette  remove  5  c.c.  of  the 
ethereal  extract,  evaporate  it  to  dryness,  boil  the  residue  with  10  c.c.  of  40  per  cent 
alcohol,  and  cool  the  alcoholic  solution.  Make  the  volume  10  c.c,  filter  through 
a  dry  paper  if  necessary  to  remove  fat.  and  to  5  c.c.  of  the  filtrate,  which  represents 
2  c.c.  of  milk,  add  2  c.c.  of  a  2  per  cent  solution  of  ferric  chloride.  The  production 
of  a  purple  or  violet  color  indicates  the  presence  of  salicylic  acid. 

'  For  other  tests  see  Sherman's  Organic  .Analysis,  Second  Edition,  p.  3 78. 


324 


PHYSIOLOGICAL   CHEMISTRY 


This  test  may  form  the  basis  of  a  quantitative  method  by  diluting  the  final 
solution  to  so  c.c.  and  comparing  this  with  standard  solutions  of  salicylic  acid. 
The  colorimetric  comparisons  may  be  made  in  a  Duboscq  colorimeter. 

(c)  Hydrogen  Peroxide. — Add  2-3  drops  of  a  2  per  cent  aqueous  solution  of 
para-phenylenediamine  hydrochloride  to  10-15  c.c.  of  milk.  If  hydrogen  peroxide 
is  present  a  blue  color  will  be  produced  immediately  upon  shaking  the  mixture  or 
after  allowing  it  to  stand  for  a  few  minutes.  It  is  claimed  that  hydrogen  peroxide 
may  be  detected  by  this  test  when  present  in  the  proportion  i  :  40,000. 

{d)  Boric  Acid  and  Borates. — To  the  ash,  obtained  accord- 
ing to  the  directions  given  in  Experiment  4,  page  327,  add  2 
drops  of  dilute  hydrochloric  acid  and  i  c.c.  of  water.  Place  a 
strip  of  turmeric  paper  in  the  dish  and  after  allowing  it  to  soak 
for  about  one  minute  remove  it  and  allow  it  to  dry  in  the  air. 
The  presence  of  boric  acid  is  indicated  by  the  production  of  a 
deep  red  color  which  changes  to  green  or  blue  upon  treatment 
with  a  dilute  alkali.  This  test  is  supposed  to  show  boric  acid 
when  present  in  the  proportion  i  :  8000. 

Quantitative  Analysis  of  Milk 

1.  Specific  Gravity. — This  may  be  determined  con- 
veniently by  means  of  a  Soxhlet,  Veith,  or  Quevenne 
lactometer.  A  lactometer  reading  of  32°  denotes  a 
specific  gravity  of  1.032.  The  determination  should 
be  made  at  about  6o°F.  and  the  lactometer  reading 
corrected  by  adding  or  subtracting  0.1°  for  every  degree 
F.  above  or  below  that  temperature. 

2.  Fat. — (a)  Babcock's  Centrifugal  Method.^ — Prin- 
ciple.— The  principle  of  this  method  is  the  destruction 
of  organic  matter  other  than  fat  by  sulphuric  acid  and 
the  centrifugation  of  the  acid  solution  in  the  special 
tube  shown  in  Fig.  106  and  the  subsequent  reading  of 
the  percentage  of  fat  by  means  of  the  tube's  gradu- 
ated neck.  The  method  is  one  of  the  most  satisfac- 
tory in  common  use  and  is  accurate  to  within  0.5  per 
cent. 


5C,c.i 


Fig.  106. — Bab- 
cock  Tube. 


Procedure. — By  means  of  a  special  narrow  pipette  introduce  milk  into  the 
tube  up  to  the  5  c.c.  mark.  Now  add  sufficient  sulphxiric  acid  (sp.  gr.  1.83- 
1.834)  to  fill  the  body  of  the  tube  and  rotate  the  tube  to  secure  a  homogeneous 
acid-milk  solution.  Fill  the  neck  of  the  tube  with  an  acid-alcohol  mixture.^ 
Centrifuge  the  tube  and  contents  for  one  to  two  minutes  and  read  off  the  per- 
centage of  fat  by  means  of  the  graduated  neck  of  the  tube.     If  the  top  of  the  fat 

'  A  modification  of  this  method  for  use  with  sweetened  dairy  products,  e.g.,  ice  cream, 
and  entailing  the  use  of  a  different  type  of  centrifuge  tube  has  been  proposed  by  Halverson 
{Jour.  Ind.  and  Eng.  Chem.,  5,  403,  1913)-  ,     ,      ... 

2  This  mixture  consists  of  equal  volumes  of  amyl  alcohol  and  concentrated  hydrochloric 
acid. 


MILK 


325 


column  is  not  at  zero  it  may  be  brought  there  by  the  addition  of  water  and  a 
moment's  recentrifugation. 

In  case  very  rich  milk  ('over  5  per  cent  fat  1  is  under  examination,  it  may  be 
diluted  with  an  equal  volume  of  water  before  examination  and  the  fat  percentage 
multiplied  by  2.  In  the  examination  of  cream  it  is  customary  to  dilute  the  sample 
with  four  volumes  of  water  and  multiply  the  resultant  fat  value  by  5. 

(b)  Quantitative  Determination  of  Fat  in  Milk  by  the  Meigs'  Method  with 
Modification  and  Improved  Apparatus  by  Croll.-— The  method  as  stated  by  Dr. 
Meigs  is:  Approximately  10  c.c.  of  milk  is  care- 
fully weighed  and  transferred  to  an  ordinary 
100  c.c.  glass-stoppered  graduated  cyUnder. 
Twenty  c.c.  each  of  distilled  water  and  ether 
(0.720)  are  added,  the  ground-glass  stopper 
tightly  inserted  in  the  bottle,  and  the  whole 
shaken  vigorously  for  five  minutes.  Then  the 
bottle  is  carefully  unstoppered,  20  c.c.  95  per 
cent  alcohol  added,  the  stopper  reinserted  and 
again  shaken  for  five  minutes.  The  bottle  is 
now  placed  on  a  table  and  the  contents  will 
separate  into  two  distinct  strata,  the  upper  of 
which  contains  practically  all  the  fat.  This 
stratimi  is  carefully  removed  by  a  smaU  pipette 
and  transferred  to  a  carefully  weighed  glass 
evaporating  dish.  The  thin  ether  layer  remain- 
ing is  washed  by  the  addition  of  5  c.c.  of  ether. 
This  is  removed  by  pipetting  off.  This  wash- 
ing is  repeated  four  times.  On  each  addition 
the  sides  of  the  bottle  should  carefuUy  be 
washed  down  by  the  fresh  ether.  Finally,  the 
pipette  is  rinsed  with  a  little  ether.  The 
evaporating  dish  with  contents  is  now  placed 
on  a  safety  water-bath  and  the  ether  evapo- 
rated. The  drying  is  continued  in  a  hot-air 
oven  at  a  temperature  below  loo^C.  and  finally 
completed  in  a  desiccator  to  constant  weight. 

CroU's  modification  consists  of  subsequent 
repeated  extraction  of  the  end-product  of 
evaporation  with  absolute  ether.  The  com- 
bined extracts  are  filtered  and  the  small  filter 

paper  is  washed  repeatedly  with  absolute  ether.     The  combined  extracts  and 
washings  are  evaporated  and  dried  as  before  and  then  weighed. 

The  piece  of  apparatus  shown  in  Fig.  107,  above  was  also  devised  by  Croll 
to  do  away  with  the  use  of  the  pipette.  ^  On  closing  the  top  with  a  finger  and 
blowing  into  the  mouthpiece,  the  upper  stratum  is  forced  out  into  the  dish.  The 
bottle  is  washed  by  simply  pouring  the  ether  into  the  tube.  This  lessens  the 
possibility  of  accidental  loss. 

^  Original  paper  by  Dr.  .Arthur  V.  Meigs  in  Philadelphia  Medical  Times,  July  i,  1882. 
^  Croll:  Biochem.  Bull.,  2,  509.  1913. 

'If  desired  a  cork  with  two  tubes  may  be  substituted  for  this  somewhat  complicated 
apparatus. 


Fig.  107. — Croll's  Fat 
ajppar.\tus. 


326 


PHYSIOLOGICAL    CHEMISTRY 


The  accuracy  of  the  method  compared  with  that  of  the  Soxhlet  method, 
using  the  paper-coil  modification  and  extracting  until  fresh  portions  of  absolute 
ether  gave  no  further  trace  of  extractive  material,  is  shown  by  the  average 

difference  on  twelve  samples  of  human  milk 
being  only  0.017  per  cent  less  than  by  the 
Soxhlet  and  on  seven  samples  cow's  milk  being 
only  0.019  per  cent  less.  The  extreme  differ- 
ences in  case  of  the  hrnnan  milk  were — 0.004 
per  cent  and — 0.044  per  cent  and  in  case  of 
the  cow's  milk — 0.006  per  cent  and — 0.068  per 
cent. 

(f)  Adams'  Paper-coil  Method. — Introduce 
about  5  c.c.  of  milk  into  a  small  beaker,  quickly 
ascertain  the  weight  to  centigrams,  stand  a  fat- 
free  coU^  in  the  beaker  and  incline  the  vessel 
and  rotate  the  coil  in  order  to  hasten  the  absorp- 
tion of  the  milk.  Immediately  upon  the  com- 
plete absorption  of  the  milk  remove  the  coU  and 
again  quicklj^  ascertain  the  weight  of  the  beaker. 
The  difference  in  the  weights  of  the  beaker  at 
the  two  weighings  represents  the  quantity  of 
milk  absorbed  by  the  coil.  Dry  the  coil  care- 
fully at  a  temperature  below  loo^C.  and  extract 
it  with  ether  for  3-5  hours  in  a  Soxhlet  appa- 
ratus (Fig.  108).  Using  a 
safety  water-bath,  heat  the 
flask  containing  the  fat  to 
constant  weight  at  a  tempera- 
ture below  loo'^C. 

Calculation. — Divide    the 
weight   of   fat,   in    grams,    by 
the  weight  of  milk,  in  grams. 
The  quotient  is  the  percentage 
Fig.  108. — Soxhlet  Apparatus,  of  fat  contained   in   the  milk 

examined. 

(d)  Nephelometric  Method  of  Bloor.- — This  method  is  ex- 
actly similar  in  principle  and  procedure  to  the  method  given 
for  the  determination  of  fat  in  blood.  (See  page  295.)  One 
c.c.  of  milk  is  ordinarily  taken. 

(e)  Approximate  Determination  by  Feser's  Lactoscope. 
is  opaque  mainly  because  of  the  suspended  fat  globules  and 
therefore  by  means  of  the  estimation  of  this  opacity  we  may  obtain  data  as  to 
the  approximate  content  of  fat.  Feser's  lactoscope  (Fig.  109)  may  be  used  for 
this  purpose.  Proceed  as  follows:  By  means  of  the  graduated  pipette  accompany- 
ing the  instrument  introduce  4  c.c.  of  milk  into  the  lactoscope.  Add  water  gradually, 
shaking  after  each  addition,  and  note  the  point  at  which  the  black  lines  upon  the 
inner  white  glass  cylinder  are  distinctly  visible.     Observe  the  point  on  the  graduated 

^  Very  satisfactory  coils  are  manufactured  by  Schleicher  and  Schiill. 
^  Bloor:  J.  Am.  Chem.  Soc,  36,  1300,  1914. 


f\ 


—  I 


0^^ 


^j-ii.  Fig.  109 — Feser's 
'  Lactoscope. 


MILK 


327 


scale  of  the  lactoscope  which  is  level  with  the  surface  of  the  diluted  milk.  This 
reading  represents  the  percentage  of  fat  present  in  the  undiluted  milk.  Pure  milk 
should  contain  at  least  3  per  cent  of  fat. 

3.  Total  Solids.' — Introduce  25  grams  of  milk  into  a  weighed  fiat-bottomed 
platinum  dish-  and  quickly  ascertain  the  weight  to  milligrams.  Expel  the  major 
portion  of  the  water  by  heating  the  open  dish  on  a  water-bath  and  continue  the 
heating  in  an  air-bath  or  water  oven  at  97'  ioo°C.  until  the  weight  is  con- 
stant. (If  platinum  dishes  are  employed  this  residue  may  be  used  in  the 
determination  of  ash  according  to  the  method  described  below,  i 

Calculation. 3 — Divide  the  weight  of  the  residue,  in  grams,  by  the  weight  of 
milk  used,  in  grams.  The  quotient  is  the  percentage  of  solids  contained  in  the 
milk  examined. 

4.  Ash. — Heat  the  dry  solids  from  2-5  grams  of  milk,  obtained  according  to 
the  method  just  given,  over  a  very  low  flame ^  until  a  white  or  light  gray  ash  is 
obtained.  Cool  the  dish  in  a  desiccator  and  weigh.  fThis  ash  may  be  used  in 
testing  for  borates  according  to  directions  on  page  324.) 

5.  Proteins :  Nephelometric  Determination  of  Proteins,  Casein, 
Globulin,  and  Albumin  in  Milk.  Method  of  Kober." — Principle. — The 
proteins  are  precipitated  with  sulphosahcyhc  acid  and  the  precipitate 
estimated  nephelometricaliy  (see  discussion  of  nephelometric  methods, 
page  290J. 

Procedure. — Five  c.c.  of  milk  are  carefully  measured  into  a  250  c.c.  flask 
and  after  adding  200  c.c.  of  distilled  water  and  10  c.c.  of  decinormal  sodium 
hydroxide  solution,  water  is  added  to  the  mark  and  the  mixture  shaken.  Ten 
c.c.  are  put  with  exactly  2  c.c.  of  ether  in  a  centrifuge  tube  which  is  then  tightly 
stoppered  with  a  cork  and  vigorously  shaken.  Allow  to  separate  and  withdraw 
5  c.c.  of  the  aqueous  layer  without  contamination  with  ether.  Dilute  to  50  c.c. 
Take  10  c.c.  of  this  solution  and  add  10  c.c.  of  3  per  cent  sulphosaUcylic  acid.  A 
suspension  of  casein  is  obtained  which  can  be  matched  acciirately  with  the 
following  standard :  i  voliime  (5  c.c.)  of  a  o.oi  per  cent  casein  solution*  to  which 
is  added  2  volimies  (10  c.c.)  of  3  per  cent  siilphosahcylic  acid. 

The  protein  obtained  with  this  reagent  is  not  all  casein,  and  in  order  to  obtain 
the  exact  amount  of  casein  the  casein  is  precipitated  according  to  the  "official 

*  Shackell's  method  for  the  vacuum  desiccation  of  frozen  preparations  may  be  used 
where  great  accuracy  is  desired  (see  American  Journal  of  Physiology,  24,  325,  1909). 

^Lead  foil  dishes,  costing  only  about  one  dollar  per  gross,  make  a  very  satisfactory 
substitute  for  the  platinum  dishes. 

'  The  percentage  of  total  solids  may  be  calculated  from  the  specific  gravity  and  percentage 
of  fat  bj'  means  of  the  following  formula  which  has  been  proposed  by  Richmond: 

3  =  0.25  L  +  i. 2  F+0.14 
S  =  total  solids. 
L  =  lactometer  reading. 
F  =  fat  content. 

*  Great  care  should  be  used  in  this  ignition,  the  dish  at  no  time  being  heated  above  a 
faint  redness,  as  chlorides  may  volatilize. 

*  Kober:  /.  Am.  Chem.  Soc,  35,  1585,  1913. 

^Standard  Casein  Solution. — Dissolve  with  stirring  o.i  gram  of  casein  or  its  equivalent 
in  r  c.c.  of  o.i  N  NaOH,  add  95  c.c.  of  distilled  water,  add  2  c.c.  of  toluene,  shake  thoroughly 
and  make  up  to  100  c.c.  This  is  the  stock  solution  which  keeps  three  or  four  days  or  longer. 
The  standard  solution  is  made  up  fresh  every  day  by  making  10  c.c.  of  the  stock  solution 
up  to  100  c.c.  with  water.  The  standard  is  controlled  bj-  total  nitrogen  estimations  using 
the  factor  6.38  for  casein. 


328  PHYSIOLOGICAL   CHEMISTRY 

method"  or  the  method  of  Hart  given  below  and  the  amount  of  precipitate  ob- 
tained in  an  aliquot  portion  of  the  filtrate,  by  adding  4  volimies  of  the  reagent, 
is  determined  nephelometricaUy.  This  fraction,  for  want  of  a  better  name  called 
the  "globulin  and  albimiin  fraction,"  is  subtracted  from  the  gross  casein,  to 
give  the  amount  of  casein  precipitated  by  the  "oflBcial  method." 

The  ether  used  in  extracting  the  fat  increases  the  volume  of  the  solution  and 
hence  a  factor  allowing  for  this  must  be  used.  For  10  c.c.  of  diluted  milk  and 
2  c.c.  of  ether  the  factor  is  0.910. 

6.  Proteins. — Introduce  a  known  weight  of  milk  (5-10  grams)  into  a  500  c.c. 
Kjeldahl  digestion  flask  and  add  20  c.c.  of  concentrated  sulphuric  acid  and  about 
0.2  gram  of  copper  sulphate.  Expel  the  major  portion  of  the  water  by  heating 
over  a  low  flame  and  finally  use  a  full  flame  and  allow  the  mixture  to  boil  one  to 
two  hours.  Complete  the  determination  according  to  the  directions  given  under 
Kjeldahl  Method,  page  483. 

Calculation. — Multiply  the  total  nitrogen  content  by  the  factor  6.37^  to  obtain 
the  protein  content  of  the  milk  examined. 

7.  Hart's  Casein  Method.- — Introduce  10.5  c.c.  of  milk  into  a  200  c.c.  Erlen- 
meyer  flask  and  add  75  c.c.  of  distilled  water  and  1-1.5  c.c.  of  10  per  cent  acetic 
acid.^  Mix  the  contents  by  giving  the  flask  a  vigorous  rotary  motion.  The 
precipitated  casein  is  now  filtered  off  upon  a  9-1 1  cm.  filter  paper.*  Wash  out 
the  adsorbed  and  loosely  combined  acetic  acid  by  means  of  cold  water.  Con- 
tinue the  washing  of  both  the  casein  on  the  filter  and  that  adhering  to  the  flask, 
until  the  wash  water  has  reached  a  volume  of  at  least  250  c.c. 

Now  return  the  precipitate  and  paper  to  the  original  Erlenmeyer  flask,  add 
75-80  c.c.  of  neutral  (carbon  dioxide-free)  water,  10  c.c.  of  N/io  potassimn  hy- 
droxide and  a  few  drops  of  phenolphthalein.  Stopper  the  flask  and  shake  it 
vigorously,  by  hand  or  machine,  until  the  casein  has  been  brought  into  solution.^ 
Rinse  the  stopper  with  neutral  (carbon  dioxide-free)  water  and  titrate  the  alka- 
line casein  solution  at  once  with  N/io  hydrochloric  acid  until  there  is  a  dis- 
appearance of  all  red  color.  ^ 

Calculation. — Subtract  the  corrected®  acid  reading  from  the  10  c.c.  of  alkali 
used.  The  difference  is  the  percentage  of  casein  in  the  milk.  For  example,  if 
it  takes  6.7  c.c.  of  N  10  hydrochloric  acid  to  titrate  the  alkaline  solution  to  the 
end  point  and  the  check  test  was  equivalent  to  20  c.c.  N/io  acid  the  casein  value 
would  be  obtained  as  follows : 

10  — (6.7+0.2)  =  3.1  per  cent  casein 

^  The  usual  factor  employed  for  the  calculation  of  protein  from  the  nitrogen  content  is 
6.25  and  is  based  on  the  assumption  that  proteins  contain  on  the  average  16  per  cent  of 
nitrogen.  This  special  factor  of  6.37  is  used  to  calculate  the  protein  content  from  the  total 
nitrogen,  since  the  principal  protein  constituents  of  milk,  i.e.,  casein  and  ladalbumin, 
contain  about  15.7  per  cent  of  nitrogen. 

*Hart:  Jour.  Biol.  Chem.,  6,  445,  1909. 

•  In  general  1.5  c.c.  of  acetic  acid  gives  a  clear  solution  which  filters  nicely  but  occasion- 
ally, when  the  milk  has  a  low  casein  value  it  is  advisable  to  use  less  acetic  acid. 

•  The  process  of  filtration  may  be  retarded  through  the  packing  of  the  casein  mass 
upon  the  filter  paper.  In  this  case  conduct  a  fine  stream  of  cold  water  against  the  upper 
point  of  contact  of  filter  paper  and  casein.  By  this  means  the  casein  precipitate  is  loosened 
and  gathers  in  the  apex  of  the  filter.  This  procedure  is  very  essential.  It  is  not  necessary 
to^remove  the  casein  which  adheres  to  the  interior  of  the  flask. 

'  Solution  is  indicated  by  the  disappearance  of  the  white  casein  particles  which  would 
otherwise  settle  to  the  bottom  of  the  flask. 

•  A  check  test  should  be  run  parallel  with  the  entire  determination.  Even  with  special 
precautions  as  to  neutrality,  it  is  generally  found  that  an  acid  check  of  0.2-0.3  wUl  be 
obtained.     This  check  titration  should  be  added  to  the  volume  of  acid  used  in  titration. 


MILK  329 

8.  Casein. — Mix  about  20  grams  of  mUk  with  40  c.c.  of  a  saturated  solution 
of  magnesuim  sulphate  and  add  the  salt  in  substance  until  no  more  will  dissolve. 
The  precipitate  consists  of  casein  admixed  with  a  little  fat  and  lacto-globulin. 
Filter  off  the  precipitate,  wash  it  thoroughly  with  a  saturated  solution  of  magnesium 
sulphate,^  transfer  the  filter  paper  and  precipitate  to  a  Kjeldahl  digestion  flask,  and 
determine  the  nitrogen  content  according  to  the  directions  given  in  a  previous 
experiment  (6). 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to  obtain  the  casein 
content. 

9.  Lactalbumin. — To  the  filtrate  and  washings  from  the  determination  of 
casein,  in  Experiment  8,  add  Almen's-  reagent  until  no  more  precipitate  forms. 
Filter  off  the  precipitate  and  determine  the  nitrogen  content  according  to  the  direc- 
tions given  under  Proteins,  page  328. 

Calculation. — Multiply  the  total  nitrogen  by  the  factor  6.37  to  obtain  the  lactal- 
bumin content. 

10.  Lactose. — To  about  350  c.c.  of  water  in  a  beaker  add  20  grams  of  milk, 
mix  thoroughly,  acidify  the  fluid  with  about  2  c.c.  of  lo  per  cent  acetic  acid  and 
stir  the  acidified  mixture  continuously  until  a  flocculent  precipitate  forms.  At 
this  point  the  reaction  should  be  distinctly  acid  to  litmus.  Heat  the  solution  to 
boiling  for  one-half  hour,  filter,  rinse  the  beaker  thoroughly,  and  wash  the  pre- 
cipitated proteins  and  the  adherent  fat  with  hot  water.  Combine  the  filtrate 
and  wash  water  and  concentrate  the  mixture  to  about  150  c.c.  Cool  the  solution 
and  dilute  it  to  200  c.c.  in  a  voliunetric  flask.  Titrate  this  sugar  solution  accord- 
ing to  directions  given  under  Fehling's  Method,  page  523  or  Benedict's  Method, 
page  522. 

Myers'  recommends  the  following  procedure  for  the  determination  of  lactose 
in  milk.  One  part  of  milk  is  mixed  with  an  equal  volume  of  phosphotungstic 
acid  solution  ''yo.o  grams  acid  and  200  c.c.  cone.  HCl  in  i  Uter  of  water  1  and  2-3 
parts  of  water.  Mix  well,  filter  until  clear,  and  titrate  the  clear  filtrate  against 
Benedict's  solution  (2$  c.c.  reducing  67  mg.  of  lactose.) 

The  milk  may  also  be  clarified  for  the  lactose  determination  by  means  of 
altimintun  hydroxide ''  or  dialyzed  iron.  *  The  dialyzed  iron  procedure  is  as  follows: 
Dilute  10  gm.  of  milk  to  25  c.c.  and  add  about  3  c.c.  of  10  per  cent  colloidal  iron 
solution  adding  the  last  portion  drop  by  drop  to  determine  the  exact  amoxmt 
necessary.  Filter  and  wash  with  water  to  make  the  clear  filtrate  100  c.c.  Titrate 
using  Benedict's  method  (see  page  522). 

The  preparation  of  aluminiiun  hydroxide  cream  and  its  use  in  protein  re- 
moval are  described  under  Nitrogen  Partition,  p.  485,  Chapter  XXVI. 

Calculation. — Make  the  calculation  according  to  directions  given  imder 
Fehling's  Method,  page  523,  bearing  in  mind  that  10  c.c.  of  Fehling's  solution 
is  completely  reduced  by  0.0676  gram  of  lactose.* 

^  Preserve  the  filtrate  and  washings  for  the  determination  of  lactalbumin  (Expt.  9). 
^Alm6n's  reagent  may  be  prepared  by  dissolving  5  grams  of  tannic  acid  in  240  c.c.  of 
50  per  cent  alcohol  and  adding  10  c.c.  of  25  per  cent  acetic  acid. 

*  Myers:  Miinch.  med.  Woch.,  59,  1494,  1912. 

*  Welker  and  Marsh:  /.  Am.  Chem.  Soc,  35,  823,  1913. 
*Hill:  Jour.  Biol.  Chem.,  20,  175,  1915. 

'  In  case  Benedict's  method  is  used  it  should  be  remembered  that  25  c.c.  of  the  reagent 
is  reduced  by  0.067  gram  of  lactose. 


CHAPTER  XVIII 
EPITHELIAL  AND  CONNECTIVE  TISSUES 

EPITHELIAL  TISSUE  (KERATIN) 

The  albuminoid  keratin  constitutes  the  major  portion  of  hair,  horn, 
hoof,  feathers,  nails,  and  the  epidermal  layer  of  the  skin.  There  is  a 
group  of  keratins  the  members  of  which  possess  very  similar  properties. 
The  keratins  as  a  group  are  insoluble  in  the  usual  protein  solvents  and 
are  not  acted  upon  by  the  gastric  or  pancreatic  juices.  They  all  re- 
spond to  the  xanthoproteic  and  Millon  reactions  and  are  characterized 
by  containing  large  amounts  of  sulphur.  Keratin  from  any  of  its 
sources  may  be  prepared  in  a  pure  form  by  treatment,  in  sequence,  with 
artificial  gastric  juice,  artificial  pancreatic  juice,  boiling  alcohol,  and 
boiling  ether,  from  twenty-four  to  forty-eight  hours  being  devoted 
to  each  process. 

The  percentage  composition  of  some  typical  keratins  is  given  in  the 
following  table: 


Source 

Percentage  composition 

S 

N 

C 

! 
H 

0 

Nailsi 

2  .  80 

17-51 

51.00 

6.94 

21.75  [ 

Honi2 

3-20 

50.86 

6.94  ' 



w 

e 
1 

3 

X 

Indian 

4.82 

15.40 

44.06 

6-53 

29.19 

Japanese  . . 

4-96 

14.64 

42.99 

5-91 

31-50 

Negro .... 

4.84 

14.90 

43-85 

6-37 

30.04 

Caucasian 

(adults) 

'     C.22 

15-79 

44-49 

6.44 

28.66 

Caucasian  (children) 

4-93 

1 

14-58 

43-23 

6.46 

30.80 

The  composition  of  human  hair  is  influenced  by  its  color  and  by  the 
race,  sex,  age  and  purity  of  breeding  of  the  individual.^     It  may  be  dif- 

^  Mulder:  Versuch  einer  allgetn.  physiol.  Chem.,  Braunschweig,  1844-51. 
^  Horbaczewski:  Ladenburg's  Handworterhtich  d.  Chem.,  3. 
^  Rutherford  and  Hawk:  Jour.  Biol.  Chem.,  3,  459,  1907. 

330 


EPITHELIAL   AND    CONNECTR'E    TISSUES  33 1 

ferentiated  from  all  other  animal  hair  or  wool  by  its  high  content  of 
cystine.     Human  hair  may  yield  nearly  12  per  cent  of  this  amino-acid.^ 

Experiments  on  Epithelial  Tissue 

Keratin 

Horn  shavings  or  nail  parings  may  be  used  in  the  experiments  which 
follow : 

1.  Solubility. — Test  the  solubility  of  keratin  in  water,  dilute  and  concentrated 
acid  and  alkali. 

2.  Millon's  Reaction. 

3.  Xanthoproteic  Reaction. 

5.  GlycxyUc  Acid  Reaction  (Hopkins-Cole). 

6.  Test  for  Unoxidized  Sulphur. 

CONNECTIVE  TISSUE 

I.  WHITE  FIBROUS  TISSUE 

The  principal  solid  constituent  of  white  fibrous  connective  tissue 
is  the  albuminoid  collagen.  This  body  is  also  found  in  smaller  per- 
centage in  cartilage,  bone,  and  ligament,  but  the  collagen  from  the 
various  sources  is  not  identical  in  composition.  In  common  with  the 
keratins,  collagen  is  insoluble  in  the  usual  protein  solvents.  It  differs 
from  keratin  in  containing  less  sulphur.  One  of  the  chief  character- 
istics of  collagen  is,  according  to  Hofmeister,  the  property  of  being 
hydrolyzed  by  boiling  acid  or  water  with  the  formation  of  gelatin. 
Emmett  and  Gies-  claim  that  under  these  conditions  there  is  an  intra- 
molecular rearrangement  of  collagen  and  the  resultant  gelatin  is  conse- 
quently not  the  product  of  hydrolysis.  The  liberation  of  ammonia  from 
the  collagen  during  the  process  apparently  confirms  this  view.  Collagen 
gives  Millon's  reaction  as  well  as  the  xanthoproteic  and  biuret  tests. 

The  form  of  white  fibrous  tissue  most  satisfactory  for  general  ex- 
periments is  the  tendo  Achillis  of  the  ox.  According  to  Buerger  and 
Gies'"^  the  fresh  tissue  has  the  following  composition: 

Water 62.87% 

Solids 37  ■  13 

Inorganic  matter 0.47 

Organic  matter 36.66 

Fattj'  substance  (ether-soluble) i  .04 

Coagulable  protein 0.22 

Mucoid 1.28 

Elastin 1.63 

Collagen 31-59 

E.xtractives,  etc 0.90 

'  Buchtala:  Zeil.  physiol.  Clioii.,  85,  24O,  1913. 

^Emmett  and  Gies:  Jour.  Biol,  cheni.,  3,  .\xxiii  (Proceedings),  1907. 

'  Buerger  and  Bies:  .-Iw.  Jour.  Physiol.,  6,  219,  1901. 


332  PHYSIOLOGICAL   CHEMISTRY 

The  mucoid  just  mentioned  is  called  tendomiicoid^  and  is  a  glyco- 
protein. It  possesses  properties  similar  to  those  of  other  connective- 
tissue  mucoids,  e.g.,  osseomucoid  and  chondromucoid. 

Gelatin,  the  body  which  results  from  the  hydrolysis  of  collagen 
(see  statement  of  Emmett  and  Gies  p.  331),  is  sometimes  classed  as  an 
albuminoid  (see  Chapter  V).  It  responds  to  nearly  all  the  protein 
tests.  It  differs  from  the  keratins  and  collagen  in  being  easily  digested 
and  absorbed.  Gelatin  is  not  a  satisfactory  substitute  for  the  protein 
constituents  of  a  normal  diet,  however,  since  a  certain  portion  of  its 
nitrogen  is  not  available  for  the  uses  of  the  organism.  Gelatin  from 
cartilage  differs  from  gelatin  from  other  sources  in  containing  a  lower 
percentage  of  nitrogen.  Tyrosine  and  tryptophane  are  not  numbered 
among  the  decomposition  products  of  gelatin,  hence  it  does  not  respond 
to  ]Millon's  reaction  or  the  glyoxylic  acid  reaction.  Cystine  is  also 
absent. 

Experiments  on  White  Fibrous  Tissue 

The  tendo  Achillis  of  the  ox  may  be  taken  as  a  satisfactory  type  of 
the  white  fibrous  connective  tissue. 

I.  Preparation  of  Tendomucoid. — Dissect  away  the  fascia  from  about  the 
tendon  and  cut  the  clean  tendon  into  small  pieces.  Wash  the  pieces  in  running 
water,  subjecting  them  to  pressure  in  order  to  remove  as  much  as  possible  of 
the  soluble  protein  and  inorganic  salts.  This  washing  is  very  important.  Trans- 
fer the  washed  pieces  of  tendon  to  a  flask  and  add  300  c.c.  of  half -saturated  lime 
water.-  Shake  the  flask  at  intervals  of  twenty-four  hours.  Filter  off  the  pieces 
of  tendon  and  precipitate  the  mucoid  with  dilute  hydrochloric  acid.  Allow  the 
mucoid  precipitate  to  settle,  decant  the  supernatant  fluid  and  filter  the  remainder. 
Test  the  mucoid  as  follows : 

(a)  SolubiUty. — Try  the  solubility  in  water,  sodixmi  choride,  dilute  and  con- 
centrated acid  and  alkaU. 

(b)  Biuret  Test. — First  dissolve  the  mucoid  in  potassium  hydroxide  solution 
and  then  add  a  dilute  solution  of  copper  sulphate. 

(c)  Test  for  Unoxidized  Sulphur. 

(d)  Hydrolysis  of  Tendomucoid. — Place  the  remainder  of  the  mucoid  in  a 
small  beaker,  add  about  30  c.c.  of  water  and  2  c.c.  of  dilute  hydrochloric  acid 
and  boil  until  the  solution  becomes  dark  brown.  Cool  the  solution,  neutralize 
it  with  concentrated  potassium  hydroxide,  and  test  by  Fehling's  test.  With  a 
reduction  of  Fehling's  solution  and  a  positive  biuret  test  what  do  you  conclude 
regarding  the  nature  of  tendomucoid? 

•  2.  Collagen.— This  substance  is  present  in  the  tendon  to  the  extent  of 
about  32  per  cent.  Therefore  in  making  the  following  tests  upon  the  pieces  of 
tendon  from  which  the  mucoid,  soluble  protein,  and  inorganic  salts  were  removed 
in  the  last  experiment,  we  may  consider  the  tests  as  being  made  upon  collagen. 

'  Cutter  and  Gies:  Am.  Jour.  Physiol.,  6,  155,  1901. 

2  Made  by  mixing  equal  volumes  of  saturated  lime  water  and  water  from  the  faucet. 


EPITHELIAL   AND   CONNECTIVE   TISSUES  ^^T, 

(a)  Solubility. — Cut  the  collagen  into  very  fine  pieces  and  try  its  solubility 
in  water  and  dilute  and  concentrated  acid  and  alkali. 

(b)  Millon's  Reaction. 

(c)  Biuret  Test. 

(d)  Xanthoproteic  Reaction. 

(e)  GlyoxyUc  Acid  Reaction  THopkins-Colej. 

(f )  Test  for  Unoxidized  Sulphur. — Take  a  large  piece  of  collagen  in  a  test- 
tube  and  add  about  5  c.c.  of  potassium  hydroxide  solution.  Heat  until  the 
collagen  is  partly  decomposed,  then  add  1-2  drops  of  lead  acetate  and  again 
heat  to  boiling. 

(g)  Formation  of  Gelatin  from  Collagen. — Transfer  the  remainder  of  the 
pieces  of  collagen  to  a  casserole,  fill  the  vessel  about  two-thirds  full  of  water 
and  boil  for  several  hours,  adding  water  at  intervals  as  needed.  By  this  means 
the  coUagen  is  transformed  and  a  body  known  as  gelatin  is  produced  'see  page 
322). 

3.  Gelatin. — On  the  gelatin  formed  from  the  transformation  of  collagen 
in  the  above  experiment  (gj,  or  on  gelatin  furnished  by  the  instructor  make  the 
following  tests : 

(a)  Solubihty. — Try  the  solubihty  in  the  ordinary  solvents  (see  page  21) 
and  in  hot  water. 

(bj  Millon's  Reaction. 

(c)  Glyoxylic  Acid  Reaction  rHopkins-Colei. — Conduct  this  test  according 
to  the  modification  given  on  page  107. 

(dj  Test  for  Unoxidized  Sulphur. 

Make  the  following  tests  upon  a  solution  of  gelatin  in  hot  water : 

(a)  Precipitation  by  Mineral  Acids. — Is  it  precipitated  by  strong  mineral 
acids  such  as  concentrated  hydrochloric  acid? 

(b)  Salting-out  Experiment. — Saturate  a  Uttle  of  the  solution  with  soUd 
ammonium  sulphate.  Is  the  gelatin  precipitated?  Repeat  the  experiment 
with  sodiimi  chloride.     What  is  the  result? 

(cj  Precipitation  by  Metallic  Salts. — Is  it  precipitated  by  metallic  salts  such 
as  copper  sulphate,  mercuric  chloride,  and  lead  acetate? 
(dj  Coagulation  Test. — Does  it  coagulate  upon  boiling? 

(e)  Precipitation  by  Alkaloidal  Reagents. ^Is  it  precipitated  by  such  reagents 
as  picric  acid,  tannic  acid,  and  trichloracetic  acid? 

(f)  Biuret  Test. — Does  it  respond  to  the  biuret  test? 

(g)  Bardach's  Reaction. — Does  it  yield  the  typical  crystals  of  this  reaction? 
(See  page  loi.j 

(h)  Precipitation  by  Alcohol. — Fill  a  test-tube  one-half  full  of  95  per  cent 
alcohol  and  pour  in  a  small  amount  of  concentrated  gelatin  solution.  Do  you 
get  a  precipitate?  How  would  you  prepare  pure  gelatin  from  the  tendo  Achillis 
of  the  ox? 

II.  YELLOW  ELASTIC  TISSUE  (ELASTIN) 

The  ligamentum  nucha  of  the  ox  may  be  taken  as  a  satisfactory  t\pe 
of  the  yellow  elastic  connective  tissue.  The  principal  solid  con- 
stituent of  this  tissue  is  elastin,  a  member  of  the  albuminoid  group. 


334  PHYSIOLOGICAL    CHEMISTRY 

In  common  with  the  keratins  and  collagen,  elastin  is  an  insoluble  body 
and  gives  the  protein  color  reactions.  It  differs  from  keratin  prin- 
cipally in  the  fact  that  it  may  be  digested  by  enzymes  and  that  it 
contains  a  very  small  amount  of  sulphur. 

It  has  been  demonstrated  that  elastin  has  the  property  of  adsorb- 
ing pepsin  from  the  gastric  juice  and  thus  protecting  it  so  the  enzyme 
can  function  later  in  the  intestine^  (see  Chapter  on  Gastric  Digestion). 

Yellow  elastic  tissue  also  contains  mucoid  and  collagen  but  these  are 
present  in  much  smaller  amount  than  in  white  fibrous  tissue,  as  may  be 
seen  from  the  following  percentage  composition  of  the  fresh  ligamentum 
nuchcB  of  the  ox  as  determined  by  Vandegrift  and  Gies.^ 

Water 57-57% 

Solids 42.43 

Inorganic  matter 0.47 

Organic  matter. 41 .  96 

Fatty  substance  (ether-soluble) 1.12 

Coagulable  protein 0.62 

Mucoid 0.53 

Elastin 31-67 

Collagen 7-23 

Extractives,  etc o .  80 

Experiments  on  Elastin 

1.  Preparation  of  Elastin  (Richards  and  Gies).^ — Cut  the  ligament  into  fine 
strips,  run  it  through  a  meat  chopper  and  wash  the  finely  divided  material  in  cold, 
running  water  for  24-48  hours.  Add  an  excess  of  half-saturated  lime  water, 
(see  note  at  the  bottom  of  page  332)  and  allow  the  hashed  ligament  to  extract 
for  48-72  hours.  Decant  the  lime  water,  remove  all  traces  of  alkali  by  washing 
in  water  and  then  boil  in  water  with  repeated  renewals  until  only  traces  of  protein 
material  can  be  detected  in  the  wash  water.  Decant  the  fluid  and  boil  the  liga- 
ment in  10  per  cent  acetic  acid  for  a  few  hours.  Treat  the  pieces  with  5  per  cent 
hydrochloric  acid  at  room  temperature  for  a  similar  period,  extract  again  in 
hot  acetic  acid  and  in  cold  hydrochloric  acid.  Wash  out  traces  of  acid  by  means 
of  water  and  then  thoroughly  dehydrate  by  boihng  alcohol  and  boihng  ether 
in  turn.     Dry  in  an  air-bath  and  grind  to  a  powder  in  a  mortar. 

2.  Solubility. — Try  the  solubility  of  the  finely  divided  elastin,  prepared  by 
yourself  or  furnished  by  the  instructor,  in  the  ordinary  solvents  (see  page  21). 
How  does  its  solubility  compare  with  that  of  collagen? 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Biuret  Test. 

6.  Glyoxylic  Acid  Reaction  (Hopkins-Cole). — Conduct  this  test  according  to 
the  modification  given  on  page  107. 

7.  Test  for  Unoxidized  Sulphur. 

^  Abderhaldcn  and  Meyer:  Zeit.  physiol.  Chem.,  74,  67,  191 1. 
-  Vandegrift  and  Gies:  Am.  Jour.  Physiol.,  5,  287,  1901. 
^  Richards  and  Gies:  Am.  Jour.  Physiol.,  7,  93,  1902. 


EPITHELIAL   AND    CONNECTIVE    TISSUES  335 

III.  CARTILAGE 

The  principal  solid  constituents  of  the  matrix  of  cartilaginous  tissue 
are  chondromucoid,  chondroitin-sulphuric  acid,  chondroalbumoid  and 
collagen.  Chondromucoid  differs  from  the  mucoids  isolated  from 
other  connective  tissues  in  the  large  amount  of  chondroitin-sulphuric 
acid  obtained  upon  decomposition.  Besides  being  an  important 
constituent  of  all  forms  of  cartilage,  chondroitin-sulphuric  acid  has 
been  found  in  bone,  ligament,  the  mucosa  of  the  pig's  stomach,  the 
kidney  of  the  ox,  the  inner  coats  of  large  arteries  and  in  human  urine. 
It  may  be  decomposed  through  the  action  of  acid  and  yields  a  nitrogenous 
body  known  as  chondroitin  and  later  this  body  yields  chondrosin. 
Chondrosin  is  also  a  nitrogenous  body  and  has  the  power  of  reducing 
Fehling's  solution  more  strongly  than  dextrose.  Levene  and  La  Forge^ 
claim  the  reducing  action  of  chondrosin  to  be  due  to  an  hexosamine 
isomeric  with  glucosamine.  Sulphuric  acid  is  a  by-product  in  the  forma- 
tion of  chondroitin,  and  acetic  acid  is  a  by-product  in  the  formation  of 
chondrosin. 

Chondroalbumoid  is  similar  in  some  respects  to  elastin  and  keratin. 
It  differs  from  keratin  in  being  soluble  in  gastric  juice  and  in  containing 
considerably  less  sulphur  than  any  member  of  the  keratin  group.  It 
gives  the  usual  protein  color  reactions. 

Experiments  on  Cartilage 

1.  Preparation  of  the  Cartilage. — Boil  the  trachea  of  an  ox  in  water  until 
the  cartilage  rings  may  be  completely  freed  from  the  surrounding  tissue.  Use 
the  cartilage  so  obtained  in  the  following  experiments : 

2.  Solubility. — Cut  one  of  the  rings  into  very  small  pieces  and  try  the  solubiUty 
of  the  cartilage  in  water  and  dilute  and  concentrated  acid  and  alkali. 

3.  Millon's  Reaction. 

4.  Xanthoproteic  Reaction. 

5.  Glyoxyhc  Acid  Reaction  (Hopkins-Cole). — Conduct  this  test  according  to 
the  modification  given  on  page  107. 

6.  Test  for  Unoxidized  Sulphur. 

7.  Preparation  of  Cartilage  Gelatin.^ — Cut  the  remaining  cartilage  rings 
into  small  pieces,  place  them  in  a  casserole  with  water  and  boil  for  several  hours. 
Filter  while  the  solution  is  still  hot.  Observe  that  the  filtrate  soon  becomes 
more  or  less  soUd.  What  is  the  reason  for  this?  Bring  a  portion  of  the  material 
into  solution  by  heat  and  try  the  following  tests : 

(a)  Biuret  Test. 

(b)  Bardach's  Reaction. 

(c)  Test  for  Unoxidized  Sulphur. 

(d)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few  drops  of  barium 
chloride.     Do  you  get  a  precipitate,  and  if  so  to  what  is  the  precipitate  due? 

^Levene  and  La  Forge:  Proc.  Soc.  cp.  Biol,  and  Med.,  ii,  124,  1914. 


336 


PHYSIOLOGICAL   CHEMISTRY 


(e)  To  about  5  c.c.  of  the  solution  in  a  test-tube  add  a  few  drops  of  dilute 
hydrochloric  acid  and  boil  for  a  few  moments.  Now  add  a  little  bariimi  chloride 
to  this  solution.  Is  the  precipitate  any  larger  than  that  obtained  in  the  preceding 
expeiment?    Why? 

(f)  To  the  remainder  of  the  solution  add  a  Uttle  dilute  hydrochloric  acid 
and  boil  for  a  few  moments.  Cool  the  solution,  neutraUze  with  solid  potassimn 
hydroxide,  and  try  FehUng's  test.     Explain  the  result. 


IV.  OSSEOUS  TISSUE 

Of  the  solids  of  bone  about  equal  parts  are  organic  and  inorganic 
matter.  The  organic  portion,  called  ossein,  may  be  obtained  by  re- 
moving the  inorganic  salts  through  the  medium  of  dilute  acid.  Ossein 
is  practically  the  same  body  which  is  termed  collagen  in  the  other 
connective  tissues,  and  in  common  with  collagen  yields  gelatin  upon 
being  boiled  with  dilute  mineral  acid. 

In  common  with  the  other  connective  tissues  bone  contains  a 
mucoid  and  an  albuminoid.  Because  of  their  origin  these  bodies  are 
called  osseomucoid  and  osseoalhumoid.  Osseomucoid,  when  boiled  with 
hydrochloric  acid,  yields  sulphuric  acid  and  a  substance  capable  of 
reducing  Fehling's  solution.  The  composition  of  osseomucoid  is  very 
similar  to  that  of  tendomucoid  and  chondromucoid  (see  page  113). 

The  inorganic  basis  of  the  dry,  fat-free  bone  is  a  chemical  substance, 
not  a  mixture.  This  fact  is  indicated  by  the  uniform  composition  of 
the  bones  of  fasting  animals  as  well  as  by  the  definite  relationship  exist- 
ing between  the  elements  present.  Bones  of  normal  and  fasting  animals 
of  the  same  species  present  no  profound  differences  in  percentage  com- 
position. The  percentage  composition  of  the  dry,  fat-free  femurs  of  two 
dogs^  after  the  animals  had  fasted  for  104  and  14  days  respectively  was 
as  follows: 


Dog  No. 

Length  of  fast 

Ash  ■ 

N 

CaO 

MgO 

P2O5 

I. 

104  days 

1 

61.50 

4.6 

33-3 

0.8 

12.80 

2. 

14  days 

61.65 

4.1 

33-1 

0.9 

12.90 

The  marked  uniformity  in  composition  notwithstanding  the  wide 
variation  in  the  fasting  periods  is  significant.  The  tensile  strength  of 
the  femur  of  the  dog  has  been  found  to  be  at  least  25,000  pounds  to  the 
square  inch^  whereas  that  of  oak  is  10,000  and  that  of  cast  iron  20,000 
pounds  to  the  square  inch. 

^  Johnston  and  Hawk :  Unpublished  data.  For  data  on  a  1 1 7-day  fast  by  dog  No.  i,  see 
Howe,  Mattill  and  Hawk:  Jour.  Biol.  Chem.,  11,  103,  1912. 


epithelial  and  connective  tissues  337 

Experiment  on  Osseous  Tissue 

The  percentage  composition  of  normal  human  bone  and  of  bone  from 
a  case  of  osteomalacia  is  given  in  the  following  table  :^ 


Constituent 

Kind  of  bone 

Normal       i  Osteomalacia 

Calcium  (CaO) 

28.85  '  15-44 
0.14                  0.57 

19.55  12.01 
0-14                  0.55 

Magnesium  (MgO) 

Phosphorus  (P2O5) 

Sulphur  (S) 

Qualitative  Analysis  of  Bone  Ash. — Take  i  gram  of  bone  ash  in  a  small 
beaker  and  add  a  little  dilute  nitric  acid.  What  does  the  effervescence  indicate? 
Stir  thoroughly  and  when  the  major  portion  of  the  ash  is  dissolved  add  an  equal 
volume  of  water  and  filter.  To  the  acid  filtrate  add  ammonium  hydroxide  to 
alkaline  reaction.  A  heavy  white  precipitate  of  phosphates  results.  (What 
phosphates  are  precipitated  here  by  the  ammonia?)  Filter  and  test  the  filtrate 
for  chlorides,  sulphates,  phosphates,  and  calciimi.  Add  dilute  acetic  acid  to 
the  precipitate  on  the  paper  and  test  a  Httie  of  this  filtrate  for  calcium  and  phos- 
phates. Heat  the  remainder  of  the  filtrate  to  boiUng  and  add  (NH4)2C03  and 
NH4CI  slowly  to  this  hot  solution  as  long  as  a  precipitate  forms.  Filter  off  the 
precipitate  of  CaCOs  and  wash  with  hot  water  until  free  from  alkaU.-  Add  a 
solution  of  Na2HP04,  make  strongly  alkaline  with  NH4OH,  and  note  the  forma- 
tion of  a  white  precipitate  of  ammonium  magnesivim  phosphate  (NH4MgP04) 
Examine  the  crystals  under  the  microscope  and  compare  with  those  shown  in 
Fig.  129,  page  408.  To  the  precipitate  on  the  filter  paper,  which  was  insoluble 
in  acetic  acid  add  a  littie  dilute  hydrochloric  acid  and  test  this  last  filtrate  for 
phosphates  and  iron. 

Reference  to  the  following  scheme  may  facilitate  the  analysis, 

^  McCrudden:  Jour.  Biol.  Cheni.,  7,  199,  1910. 

^  Magnesium  is  not  precipitated  here  because  of  presence  of  NH4CI. 


338 


PHYSIOLOGICAL   CHEMISTRY 
BONE  ASH. 


Add  dilute  nitric  acid,  stir  thoroughly  and  after  the  major  portion  of  the  ash  has  been 
brought  into  solution  add  a  little  distilled  water  and  filter. 


Residue  I.  Filtrate  I. 

(discard)  Add  ammonium  hydroxide  to 

alkaline  reaction  and  filter. 


Residue  n. 

Treat  on  paper  with  acetic  acid. 


Residue  HI. 

Treat  on  paper  with  hydro- 
chloric acid. 

I 
Filtrate  IV. 
Test  for: 

1.  Iron. 

2.  Phosphates. 


Filtrate  HI. 
Test  for: 

1.  Phosphates. 

2.  Calcium. 

3.  Magnesium 


V.  ADIPOSE  TISSUE 


Filtrate  11. 

Test  for: 

1.  Chlorides. 

2.  Sulphates. 

3.  Phosphates. 

4.  Calcium. 


Adipose  tissue  consists  almost  entirely  of  a  mixture  of  fats. 
discussion  and  experiments  see  chapter  on  Fats,  page  176. 


For 


CHAPTER  XIX 
MUSCULAR  TISSUE 

The  muscular  tissues  are  divided  physiologically  into  the  voluntary 
(striated)  and  the  involuntary  (non-striated  or  smooth).  In  the 
chemical  examination  of  muscular  tissue  the  voluntary  form  is  gener- 
ally employed.  Muscle  contains  about  25  per  cent  of  solid  matter, 
of  which  about  four-fifths  is  protein  material  and  the  remaining  one- 
fiith  extractives  and  inorganic  salts. 

The  proteins  are  the  most  important  of  the  constituents  of  muscular 
tissue.  In  the  living  muscle  we  find  two  proteins,  myosinogen  and 
para-myosinogen.  These  may  be  shown  to  be  present  in  muscle  plasma 
expressed  from  fresh  muscles.  In  common  with  the  plasma  of  the 
blood  this  muscle  plasma  has  the  power  of  coagulating,  and  the  clot 
formed  in  this  process  is  called  myosin.  According  to  Halliburton^ 
and  others  in  the  onset  of  rigor  mortis  we  have  an  indication  of  the  for- 
mation of  this  myosin  clot  within  the  body.  The  relation  between  the 
proteins  of  living  and  dead  muscle  is  represented  graphically  by  Halli- 
burton as  follows: 

Proteins  of  the  living  muscle. 


Para-myosinogen  (25%).  Myosinogen  (75%). 

Soluble  myosin. 


Myosin. 
(The  protein  of  the  muscle  clot.) 

Of  the  total  protein  content  of  living  muscle  about  75  per  cent  is 
made  up  by  the  7nyosinogen  and  the  remaining  25  per  cent  is  para- 
myosinogen. These  proteins  may  be  separated  by  subjecting  the 
muscle  plasnia  to  fractional  coagulation  in  the  usual  way.  Under 
these  conditions  the  para-myosinogen  is  found  to  coagulate  at  47°C. 
and  the  myosinogen  to  coagulate  at  56°C.  It  is  also  claimed  by  some 
investigators  that  it  is  possible  to  separate  these  two  proteins  by  the 
fractional  ammonium  sulphate  method,  but  the  possibility  of  making 

'  Halliburton:  Biochemistry  of  Muscle  and  Xervc,  1904,  p.  4. 

339 


340  PHYSIOLOGICAL   CHEMISTRY 

an  accurate  separation  by  this  method  is  somewhat  doubtful.  It  is 
well  established  that  para-myosinogen  is  a  globulin  since  it  responds  to 
certain  of  the  protein  precipitation  tests  and  is  insoluble  in  water. 
Myosinogen,  on  the  contrary,  is  not  a  typical  globulin  since  it  is 
soluble  in  water.  It  has  been  called  a  pseudo-globulin.  Myosin  pos- 
sesses the  globulin  characteristics.  It  is  insoluble  in  water  but  soluble 
in  the  other  protein  solvents  and  is  precipitated  from  its  solution  upon 
saturation  with  sodium  chloride. 

Mellanby  has  reported  observations  which  he  claims  indicate  that 
there  is  only  one  protein  in  muscle  and  that  rigor  mortis  is  due  to  the 
coagulation  of  this  protein  under  the  combined  influences  of  the  salt 
present  in  the  muscle  and  the  lactic  acid  developed  upon  the  death  of 
the  muscle.  He  further  states  that  the  disappearance  of  rigor  is  due 
to  the  fact  that  the  lactic  acid  which  is  continually  formed  brings  this 
protein  into  solution.  There  is  a  difference  of  opinion  as  to  whether 
true  rigor  ever  occurs  in  connection  with  non-striated  (smooth)^ 
muscle. 

Our  ideas  concerning  the  cause  of  rigor  have  undergone  an  im- 
portant revision  in  recent  years.  A  very  attractive  theory  has  been 
advanced  by  Meigs^  and  experimental  confirmation  has  been  accorded 
it  by  von  Fiirth  and  Lenk.^  According  to  this  theory,  rigor  has  no 
connection  with  the  coagulation  of  the  muscle  proteins  and  may  even 
be  hindered  or  prevented  by  such  coagulation.  The  cause  of  rigor, 
from  this  new  viewpoint,  lies  in  the  imbibition  of  water  by  the  muscle 
colloids.  It  is  well  known  that  colloids  possess  the  property  of  absorb- 
ing whatever  fluid  may  be  in  contact  with  them.  Moreover,  the 
capacity  of  the  colloid  for  water  is  increased  if  the  fluid  is  slightly  acid 
in  reaction.  Therefore  the  postmortem  production  of  lactic  acid 
facilitates  the  imbibition  of  muscle  fluid  by  the  muscle  colloids. 
Under  such  conditions,  the  fibers  swell,  become  rigid  and  the  condition 
known  as  rigor  mortis  results.  The  disappearance  of  rigor  is  believed 
to  be  due  to  the  coagulation  of  the  muscle  protein  through  the  agency 
of  the  accumulated  lactic  acid.  This  change  is  accompanied  by  a  re- 
lease of  the  imbibed  water  by  the  colloids,  inasmuch  as  the  capacity 
of  a  colloid  for  retaining  fluid  is  lowered  by  coagulation. 

Under  the  name  extractives  we  class  a  number  of  muscle  constituents 
which  occur  in  traces  in  the  tissue  and  may  be  extracted  by  water, 
alcohol,  or  ether.  There  are  two  classes  of  these  extractives,  the  non- 
nitrogenous  extractives  and  the  nitrogenous  extractives.     Grouped  under 

^  Saxl:  Beilriige  zur  cltemischen  Physiologic  und  Pathologie,  g,  i,  1907. 

*  Meigs:  American  Journal  of  Physiology,  26,  191,  1910. 

^  von  Fiirth  and  Lenk:  Wiener  klinische  Wochenschrift,  24,  1079,  1911. 


MUSCULAR   TISSUE  34 1 

the  non-nitrogenous  bodies  we  have  glycogen,  dextrin,  sugars,  lactic 
acid,  inosite,  C6H6(OH)6,  and/a^.  In  the  class  of  nitrogenous  extract- 
ives we  have  creatine,  creatinine,  xanthine,  hypoxanthine,  uric  acid, 
urea,  carnine,  guanine,  phosphocarnic  acid,  inosinic  acid,  carnosine, 
taurine,  carnitine,  novaine,  ignotine,  neosine,  oblitine,  carnomuscarine, 
and  methylguanidine  (see  formulas  on  pp.  127  and  346).  Not  all  of 
these  extractives  are  present  in  the  muscles  of  all  species  of  animals. 
Other  extractives  besides  those  enumerated  above  have  been  described 
and  there  are  undoubtedly  still  others  whose  presence  remains  undeter- 
mined. A  detailed  consideration  would,  however,  be  unprofitable  in 
this  place. 

Glycogen  is  an  important  constituent  of  muscle.  The  content 
of  this  polysaccharide  in  muscle  varies  and  is  markedly  decreased  by 
intense  muscular  activity.  It  is  transformed  into  sugar  and  used  as 
fuel.  The  liver  is  the  organ  which  stores  the  reserve  supply  of  glycogen 
and  transforms  it  into  glucose  which  is  passed  into  the  blood  stream 
and  so  carried  to  the  working  muscle  where  it  is  synthesized  into  gly- 
cogen. The  glycogen  thus  formed  is  then  changed  into  glucose  as  the 
working  muscle  may  need  it. 

Glycogen  is  a  polysaccharide  and  has  the  same  percentage  com- 
position as  starch  and  dextrin.  It  resembles  starch  in  forming  an  opal- 
escent solution  and  resembles  dextrin  in  being  very  soluble,  in  giving 
reddish  color  with  iodine  and  in  being  dextro-rotatory.  Glycogen  may 
be  prepared  from  muscle  by  extracting  with  boiling  water  and  then 
precipitating  the  glycogen  from  the  aqueous  solution  by  alcohol;  dilute 
or  concentrated  potassium  hydroxide  may  also  be  used  to  extract  the 
glycogen.  Glycogen  may  be  prepared  in  the  form  of  a  white,  tasteless, 
amorphous  powder.  It  is  completely  precipitated  from  its  solution 
by  saturation  with  solid  ammonium  sulphate,  but  is  not  precipitated  by 
saturation  with  sodium  chloride.  It  may  also  be  precipitated  by 
alcohol,  tannic  acid,  or  ammoniacal  basic  lead  acetate.  It  has  the 
power  of  holding  cupric  hydroxide  in  solution  in  alkaline  fluids  but 
cannot  reduce  it.  It  may  be  hydrolyzed  with  the  formation  of  glucose 
by  dilute  mineral  acids  and  is  readily  digested  by  amylolytic  enzymes. 

Mendel  and  Leavenworth  have  drawn  the  conclusion,  from  the  ex- 
amination of  embryo  pigs,  that  embryonic  structures  do  not  contain 
exceptionally  large  amounts  of  glycogen.  The  distribution  of  tlie 
glycogen  was  not  observed  to  differ  from  that  in  the  adult  animal  ex- 
cept that  the  liver  of  the  embryo  does  not  assume  its  glycogen-stor- 
ing  function  early.  They  further  draw  the  conclusion  that  the  meta- 
bolic transformations  of  glycogen  in  the  embryo  and  the  adult  are 
entirely  analogous. 


342 


PHYSIOLOGICAL    CHEMISTRY 


The  lactic  acid  occurring  in  the  muscular  tissue  of  vertebrates  is 

paralactic  or  sarcolaclic  acid,^ 

H    OH 

I        ! 
H— C— C— COOH. 

I     ! 

H    H 

The  reaction  of  an  inactive  living  muscle  is  alkaline,  but  upon  the  death 
of  the  muscle,  or  after  the  continued  activity  of  a  living  muscle,  the 
reaction  becomes  acid,  due  to  the  formation  of  lactic  acid.  There  is  a 
difference  of  opinion  regarding  the  origin  of  this  lactic  acid.  Some 
investigators  claim  it  to  arise  from  the  carbohydrates  of  the  muscle, 
while  others  ascribe  to  it  a  protein  origin.  The  strongest  evidence 
favors  a  carbohydrate  source. - 


Fig.   iio. — Creatine. 

Among  the  nitrogenous  extractives  of  muscle,  those  which  are  of  the 
most  interest  in  this  connection  are  creatine  and  the  purine  bases, 
xanthine  and  hypoxanthine.  Creatine  is  found  in  varying  amounts  in 
the  muscles  of  different  species,  the  muscles  of  birds  having  shown 
the  largest  amount.  It  has  also  been  found  in  the  blood,  the  brain,  in 
transudates  and  in  the  thyroid  gland.  Creatine  may  be  crystallized 
and  forms  colorless  rhombic  prisms  (Fig.  no)  which  are  soluble  in 
warm  water  and  practically  insoluble  in  alcohol  and  ether.  Upon 
boiling  a  solution  of  creatine  with  dilute  hydrochloric  acid  it  is  dehydro- 
lyzed  and  its  anhydride  creatinine  is  formed.     The  theory  that  the 

1  This  is  dextro-rotatory,  whereas  fermentation  lactic  acid  (<i-Mactic  acid)  is  optically 
inactive. 

''Levene  and  Meyer:  Jour.  Biol.  Chem.,  ii,  361,  1912. 


MUSCULAR   TISSUE  343 

creatine  of  ingested  meat  is  transformed  into  creatinine  and  excreted 
in  the  urine  has  been  proven  untenable  through  the  researches  of  Folin, 
Klercker,  and  Wolf  and  Shaffer.  It  is  now  known  that  under  normal 
conditions  the  ingestion  of  creatine  in  no  way  influences  the  excretion 
of  creatinine.  In  the  case  of  Eck  fistula  dogs,  however,  London  and 
Bolyarski^  found  ingested  creatine  to  increase  the  output  of  creatinine 
in  the  urine.  This  finding  is  of  importance  as  throwing  light  upon  the 
role  of  the  liver  in  creatine  and  creatinine  metabolism.  In  this  con- 
nection it  is  important  to  note  that  there  is  no  normal  excretion  of 
endogenous  (see  page  378)  creatine,  a  statement  proven  by  the  fact  that 
if  no  creatine  be  ingested  none  will  be  excreted.  Folin-  has  shown  that 
the  main  bulk  of  ingested  creatine  is  retained  in  the  body,  unless  the  diet 
contains  a  large  amount  of  protein  material.  In  fasting  the  urine 
contains  considerable  creatine,  i.e.,  120  mg.  or  more  per  day.  Under 
certain  pathological  conditions,  e.g.,  fevers,  the  urine  may  contain 
endogenous  creatine  which  is  probably  derived  from  the  catabolism 
of  muscular  tissue,  as  Benedict,  ]Mellanby,  and  Shaffer  have  suggested. 
Benedict  and  Osterberg^  believe  we  may  have  a  high  creatine  elimina- 
tion which  has  no  relation  to  the  catabolism  of  muscle. 

McCrudden'*  reports  creatine  in  the  urine  in  cases  of  infantilism 
achondroplasia  and  cretinism  the  amount  present  being  increased 
when  the  carbohydrate  ingestion  was  increased. 

It  has  been  stated  that  creatine  does  not  occur  in  non-striated 
muscle.  It  has,  however,  been  found  in  the  non-striated  muscles  of 
the  lamprey  the  lowest  form  of  vertebrates.^ 

Amberg  and  Morrill,*'  Sedgwick.'  Rose^  and  Folin^  have  shown  that 
creatine  is  a  normal  constituent  of  the  urine  of  infants  and  children 
(10-15  mg.  per  day).  Folin  explains  this  phenomenon  on  the  basis  of 
the  relatively  high  protein  intake,  whereas  Rose  believes  it  is  due  to  a 
peculiar  carbohydrate  metabolism.  ' 

Besides  being  a  normal  constituent  of  muscle,  xanthine  has  been 
found  in  the  brain,  spleen,  pancreas,  thymus,  kidneys,  testicles,  liver, 
and  in  the  urine.  It  may  be  obtained  in  crystalline  form  (Fig.  in, 
p.  344),  but  ordinarily  it  is  amorphous.  Xanthine  is  easily  soluble  in 
alkalis,  less  soluble  in  water  and  dilute  acids,  and  entirely  insoluble  in 
alcohol  and  ether. 

1  London  and  Bolyarskii:    Zeit.  phys.  chem.,  62,  465,  1909. 

*  Folin:  Hammarsten  Feslschrifl,  p.  15. 

'Benedict  and  Osterberg:  Jour.  Biol.  Chem.,  18,  195,  1914. 

*  Mc  Crudden:  Jour.  Expi.  Med.,  15,  457,  191 2. 

"Mellanby:  Jour,  of  Physiol.,  36,  472,  1908.     Wilson:  Jour.  Biol.  Chem.,  iS,  17,  1914- 
•Amberg  and  Morrill:  Jour.  Biol.  Chem.,  3,  311,  1907. 
"Sedgwick:  Jour.  Am. -Med.  Ass'n,  55,  117S,  1910. 

8  Rose:  Jour.  Biol.  Chem.,  10,  265,  191 1. 

9  Folin:  Ibid.,  11,  253,  1912. 


344 


PHYSIOLOGICAL   CHEMISTRY 


Hypoxanthine  occurs  ordinarily  in  those  tissues  and  fluids  which 
contain  xanthine.  It  has  been  found,  unaccompanied  by  xanthine,  in 
bone  marrow  and  in  milk.  Unlike  xanthine  it  may  be  easily  crystallized 
in  the  form  of  small,  colorless  needles.  It  is  readily  soluble  in  alkalis, 
acids,  and  boiling  water,  less  soluble  in  cold  water  and  practically  in- 
soluble in  alcohol  and  ether. 

The  predominating  inorganic  salt  of  muscle  is  potassium  phosphate. 
Besides  this  salt  we  have  present  chlorides  and  salts  of  sodium,  calcium, 
magnesium,  and  iron.     Sulphates  are  also  present  in  traces. 

Mendel  and  Saiki  have  made  some  interesting  observations  upon  the 
chemical  composition  of  non-striated  or  smooth  (involuntary)  mammalian 


Fig.  I II. — Xanthine. 
A  ter  the  drawings  of  Horbaczewski,  as  represented  in  Neubauer  and  Vogel.     {Ogden.) 


muscle,  such  as  the  urinary  bladder  and  the  muscular  coat  of  the  stom- 
ach of  the  pig.  Hypoxanthine  was  found  to  be  the  predominant  purine 
base  present.  Creatine  and  paralactic  acid  were  also  isolated.  These 
investigators  were  unable  to  demonstrate,  definitely,  the  presence  of 
glycogen  in  the  non-striated  muscles  studied,  but  state  that  "the 
tissues  possess  the  property  of  transforming  glycogen  in  the  char- 
acteristic enzymatic  way."  The  most  important  part  of  their  in- 
vestigation consists  in  a  rather  complete  analysis  of  the  inorganic 
constituents  of  these  muscles.  A  notable  difference  in  the  relative 
distribution  of  the  various  inorganic  constituents  was  observed,  a 
difference  which,  according  to  the  authors,  "can  be  accounted  for  in 
part  only  by  an  admixture  of  lymph."  The  comparative  composition 
of  the  inorganic  portion  of  striated  and  non-striated  muscle  and  of  blood 
serum  for  comparison  is  shown  in  the  following  table: 


MUSCULAR   TISSUE 


345 


Per  loo  parts  of  fresh  muscle 


K20lNa20Fe203 


Non-striated  muscle  (Mendel  and  Saiki)io. 08110.328 

Skeletal  muscle  (Katz) 0.3060. 210 

Blood  serum  (Abderhalden) ,0.02710.425 


CaO  !  MgO     CI     P2O6   H2O 


o.oii  0.044I0.007I0.17110.184:  80.6 
0.008  o. 01 1  0.047  0.048  0-487  72.9 
|o.oi2  0.004  0.363:0.020   91  -8 


An  interesting  comparative  study  of  the  ash  of  the  smooth  muscle  of 
the  stomach  of  the  frog  and  the  striated  muscle  from  the  same  animal 
was  very  recently  reported  by  Meigs  and  Ryan.^  Their  data  indicate 
"that  smooth  muscle  contains  somewhat  less  potassium  and  phos- 
phorus and  somewhat  more  sodium  and  chlorine  than  the  striated 
muscle  of  the  same  animal,  but  that  the  differences  in  these  respects 
between  the  two  tissues  are  not  by  any  means  so  marked  as  has  some- 
times been  supposed."  Their  average  figures  for  each  type  of  muscle 
follow : 


Per  100  parts  of  fresh  muscle 


Muscle 


K 


Na 


Fe 


Ca      Mg 


P        CI        S      SoUds   H2O 


Striated o-35o  0.054  o.oio   0.028,0.030  0.155  0.066  o.  141  20. 13    79.87 

Smooth o. 325I0. 073 jO. 0007 ■0.004I0. 013 10.137 jo. 1 20|o.  161  17.70  ,82.30 


The  preparation  from  which  the  above  data  for  smooth  muscle 
were  obtained  were  shown  by  histological  examination  to  consist  in 
large  part  of  smooth  muscle  fibers. 

Muscular  tissue  is  said  to  contain  a  reddish  pigment  called  myo- 
hematin,  which  is  a  derivative  of  hemoglobin. 

The  so-called  "fatigue  substances"  of  muscle  are  carbon  dioxide, 
paralactic  acid,  and  potassium  dihydrogen  phosphate. 

The  ordinary  commercial  "meat  extract"  is  composed  principally 
of  the  water-soluble  constituents  of  muscle  and  contains  practically 
nothing  of  nutritive  value.  The  protein  material  to  which  meat  owes 
its  value  as  an  article  of  diet  is  ordinarily  practically  all  removed  in 
the  preparation  of  the  extract.  Occasionally  some  preparations  are 
found  to  contain  proteose,  which  is  formed  from  the  meat  proteins  in 
the  process  of  preparation. 

Lusk-  has  shown  that  Liebig's  extract  is  without  influence  upon  the 


^  Meigs  and  Ryan:  Journal  of  Biological  Chemistry,  11,  401,  191 2. 
-Lusk:  Jotir.  Biol.  Chetn.,  13,  155,  1912. 


346 


PHYSIOLOGICAL   CHEMISTRY 


metabolism  (energy)  in  spite  of  the  glandular  activity  it  is  known  to 
induce. 

The  structural  formulas  of  some  of  the  nitrogenous  extractives  of 
muscle  are  as  follows: 


NH2 

HN  =  C 

I 
N.(CH3).CH2.COOH. 

Creatine,  CiHtNsOj. 

M ethyl- guanidine  acetic  actd. 

NH2 

1 

c=o 

I 

NH2 

UREA.CON2H4. 


o- 


co 


HN 
HN  =  C 


N.(CH3).CH2 

Creatinine,  C4H7N3O. 
Creatine  anhydride. 


CH2.NH2 
CH2.SO2OH 


Taurine,  C2H7NSO3. 
Amino-ethyl  sulphonic  acid. 

-CO 


(CH3)3.N 


CH2— CH.OH— CH2 

Carnitine,  CtHuNOs. 
y-trimethyloxybutyrobetaine. 

OH 


(CH3)3N 

CH2.CH2.CH2.CH(OH)2 

NOVAINE,   C7H17NO2. 

Tri-methyl  di-hydroxyhutyl  ammonium  hydroxide. 

Carnosine,  C9H14N4O3. 

Neosine,  C6H17NO2. 

Ignotine,  C9H14N4O3. 

Phosphocarnic  acid,  C10H17N3O5  or  C10H16N3O5. 

Inosinic  acid,  (HO)2.PO.O.CH2(CHOH)3.CH:(C5H3N40). 

Purine  Bodies.^ — 

HN— CO  HN— CO 


HC     C— NH 


/ 


CH 


N— C— N 

Hypoxanthine,  C6H4N40. 
6-oxypurine. 


OC     C— NH 

>CH 
HN— C— N 

Xanthine,  C6H4N4O2. 
2-6-dioxypurine. 


1  For  discussion  of  the  purine  bodies  which  are  found  as  muscle  extractive  see  Chapter 
VI  on  Nucleic  Acids. 


MUSCULAR   TISSUE  347 

HN— CO  N  =  C.NH, 

I       I  I       I 

H2N.C     C— NH  HC     C— NH 

N— C— H  N— C— N 

Guanine,  CaHsNtO.  Adenine.  dUiSi. 

2-amino~6-oxy  purine.  6 -amino  purine. 

Experiments  on  Muscular  Tissue 

I.  Experiments  on  "Living"  Muscle 

1.  Preparation  of  Muscle  Plasma  (Halliburton).-  Wash  out  the  blood  vessels 
of  a  freshly  killed  rabbit  with  0.9  per  cent  sodium  chloride.  This  can  best  be 
done  by  opening  the  abdomen  and  inserting  a  carmula  into  the  aorta.  Now 
remove  the  skin  from  the  lower  limbs,  cut  away  the  muscles  and  divide  them 
into  very  small  pieces  by  means  of  a  meat  chopper.  Transfer  the  pieces  of 
muscle  to  a  mortar  and  grind  them  with  clean  sand  and  a  Uttle  ice  cold  5  per 
cent  magnesivmi  sulphate.  Place  in  an  ice-box  over  night.  Filter  off  the  salted 
muscle  plasma  and  make  the  following  tests : 

(a)  Reaction. — Test  the  reaction  to  litmus,  phenolphthalein,  and  Congo  red. 
What  is  the  reaction  of  this  fresh  muscle  plasma? 

(b)  Fractional  Coagulation. — Place  a  little  muscle  plasma  in  a  test-tube  and 
arrange  the  apparatus  for  fractional  coagulation  as  explained  on  page  105.  Raise 
the  temperature  very  carefully  from  30°C.  and  note  any  changes  which  may  occur 
and  the  exact  temperature  at  which  such  changes  take  place.  When  the  first 
protein  (para-myosinogen)  coagulates  filter  it  ofif  and  then  heat  the  clear  filtrate 
as  before,  being  careful  to  note  the  exact  temperature  at  which  the  next  coagula- 
tion (myosinogen)  occurs.  There  will  probably  be  a  preliminary  opalescence 
in  each  case  before  the  real  coagulation  occurs.  Therefore  do  not  mistake  the 
real  coagulation-point  and  filter  at  the  wrong  time.  What  are  the  coagulation 
temperatures  of  these  two  proteins?  Which  protein  was  present  in  greater 
amount? 

(c)  Formation  of  the  Myosin  Clot. — Dilute  a  portion  of  the  plasma  with  3 
or  4  times  its  volimie  of  water  and  place  it  on  a  water-bath  or  in  an  incubator 
at  35°C.  for  several  hours.  A  typical  myosin  clot  should  form.  Note  the  muscle 
senun  surrounding  the  clot. .  Now  test  the  reaction.  Has  the  reaction  changed, 
and  if  so  to  what  is  the  change  due?  Make  a  test  for  lactic  acid.  What  do  you 
conclude? 

2.  I*reparation  of  Muscle  Plasma  (v.  Fiirth). — Remove  the  blood-free  muscles 
of  a  rabbit  as  explained  above.  Finely  divide  by  means  of  a  meat  chopper  and 
grind  in  a  mortar  with  a  little  clean  sand  and  some  0.9  per  cent  sodium  chloride. 
Wrap  portions  of  the  muscle  in  muslin  and  press  thoroughly  by  means  of  a  tincture 
press  or  lemon  squeezer.  Filter  and  make  the  tests  according  to  the  directions 
given  in  the  last  experiment. 

3.  **Fuchsin-frog"  Experiment.—  Inject  a  saturated  aqueous  solution  of  Fuch- 
sin  **S"  into  the  lymph  spaces  of  a  frog  two  or  three  times  daily  for  one  or  two 
days,  in  this  way  thoroughly  saturating  the  tissues  with  the  dye.  Pith  the  animal 
(.insert  a  heavy  wire  or  blunt  needle  through  the  occipito  atlantoid  membrane  % 
remove  the  skin  from  both  hind  legs  and  expose  the  sciatic  nerve  in  one  of  them. 


348  PHYSIOLOGICAL   CHEMISTRY 

Insert  a  small  wire  hook  through  the  jaws  of  the  frog  and  suspend  the  animal 
from  an  ordinary  clamp  or  iron  ring.  Pass  electrodes  imder  the  exposed 
sciatic  nerve,  and  after  tying  the  other  leg  to  prevent  any  muscular  movement, 
stimtilate  the  exposed  nerve  by  means  of  make  and  break  shocks  from  an  in- 
duction coil.  The  stimulated  leg  responds  by  pronounced  muscular  contrac- 
tions, whereas  the  tied  leg  remains  inactive.  Continue  the  stimulation  until 
the  muscles  are  fatigued.  The  muscular  activity  has  caused  the  production  of 
lactic  acid  and  this  in  turn  has  reacted  with  the  injected  fuchsin  to  cause  a  pink 
or  red  color  to  develop.  The  muscles  of  the  inactive  leg  still  remain  imchanged 
in  color. 

The  normal  color  of  the  Fuchsin  "S"  when  injected  was  red,  but  upon  being 
absorbed  it  became  colorless  through  the  action  of  the  alkalinity  of  the  blood. 
Upon  stimulating  the  muscles,  however,  as  above  explained,  lactic  acid  was  formed 
and  this  acid  reacted  with  the  fuchsin  and  again  produced  the  original  color  of 
the  dye. 

n.  Experiments  on  "Dead"  Muscle 

1.  Preparation  of  Myosin. — ^Take  25  grams  of  finely  divided  lean  beef 
which  has  been  carefully  washed  to  remove  blood  and  lymph  constituents  and 
place  it  in  a  beaker  with  10  per  cent  sodivmi  chloride.  Stir  occasionally  for 
several  hours.  Strain  off  the  meat  pieces  by  means  of  cheese  cloth,  filter  the 
solution  and  saturate  it  with  sodium  chloride  in  substance.  Filter  off  the  pre- 
cipitate of  myosin  and  make  the  tests  as  given  below.  This  filtration  will  pro- 
ceed very  slowly.  Myosin  collects  as  a  film  on  the  sides  of  the  filter  paper  and 
may  be  removed  and  tested  before  the  entire  volume  of  fluid  has  been  filtered. 
If  this  precipitate  remains  for  any  length  of  time  on  the  paper  in  contact  with  the 
air  it  will  become  transformed  into  the  protean  myosan.  Test  the  myosin  pre- 
cipitate as  follows : 

(a)  Solubility. — Try  its  solubility  in  water,  sodiimi  chloride,  dilute  acid  and 
alkali.     Is  myosin  an  albumin  or  a  globulin? 

(b)  Xanthoproteic  Reaction. — See  page  98. 

(c)  Coagulation  Test. — Suspend  a  Uttle  of  the  myosin  in  water  in  a  test- 
tube  and  heat  to  boiling  for  a  few  moments.  Now  remove  the  suspended  ma- 
terial and  try  its  solubility  in  10  per  cent  sodiimi  chloride.  What  property  does 
this  experiment  show  myosin  to  possess? 

Test  the  filtrate  from  the  original  myosin  precipitate  as  follows : 

(a)  Biuret  Test. — ^What  does  this  show? 

(b)  Place  a  little  of  the  solution  in  a  test-tube  and  heat  to  boiling.  At  the 
boiling-point  add  a  drop  of  dilute  acetic  acid  and  filter.  Test  this  filtrate  for 
proteose  with  picric  acid.  Is  any  proteose  present?  Saturate  another  portion 
of  the  filtrate  with  ammoniimi  sulphate  and  test  for  peptone  in  the  usual  way 
(see  page  120).    Do  you  find  any  peptone? 

From  your  experiments  on  ''living"  and  "dead"  muscle  what  are 
your  ideas  regarding  the  proteins  of  muscle? 

2.  Preparation  of  Glycogen. — Grind  a  few  oysters  or  scallops  ^  in  a  mortar 

^  Glycogen  may  also  be  prepared  from  the  liver  of  an  animal  which  has  been  fed  a  high 
carbohydrate  diet  for  1-2  days  previously.  The  best  yield  of  glycogen  can,  however,  gen- 
erally be  obtained  from  scallops.  To  secure  best  yield  of  glycogen  the  liver,  scallops  or 
oysters  should  be  fresh.  If  permitted  to  stand  some  glycogen  will  be  converted  into 
glucose. 


MUSCULAR   TISSUE  349 

with  sand.  Transfer  to  an  evaporating  dish,  add  water,  and  boil  for  20  minutes. 
Note  the  opalescence  of  the  solution.  At  the  boiUng-point  faintly  acidify  with 
acetic  acid.  Why  is  this  acid  added?  Filter,  and  divide  the  filtrate  into  two 
parts.    Test  one  part  of  the  filtrate  as  follows : 

(a)  Iodine  Test. — To  5  c.c.  of  the  solution  in  a  test-tube  add  5-10  drops  of 
iodine  solution  and  2-3  drops  of  10  per  cent  sodium  chloride.  What  do  you 
observe?  Is  this  similar  to  the  iodine  test  upon  any  other  body  with  which  we 
have  had  to  deal? 

If  difficulty  is  experienced  in  seciuing  a  satisfactory  iodine  test  proceed 
as  follows:  Make  equal  volumes  of  glycogen  solution  acid  in  reaction  with 
hydrochloric  acid.  Boil  one  solution  to  hydrolyze  the  glycogen.  Add  equal 
voliunes  of  iodine  solution  to  each  and  note  the  more  pronounced  iodine  reaction 
in  the  unhydrolyzed  solution. 

(b)  Reduction  Test. — Does  the  solution  reduce  Fehling's  solution? 

(c)  Hydrolysis  of  Glycogen. — Add  10  drops  of  concentrated  hydrochloric  acid 
to  10  c.c.  of  the  solution  and  boil  for  10  minutes.  Cool  the  solution,  neutraUze 
with  soUd  potassium  hydroxide  and  test  with  Fehling's  solution.  Does  it  still 
fail  to  reduce  FehUng's  solution?  If  you  find  a  reduction  how  can  you  prove  the 
identity  of  the  reducing  substance? 

(d)  Influence  of  SaUva. — Place  5  c.c.  of  the  solution  in  a  test-tube,  add  5 
drops  of  saUva  and  place  on  the  bath-water  at  40°C.  for  10  minutes.  Does  this 
now  reduce  Fehhng's  solution? 

To  the  second  part  of  the  glycogen  filtrate  add  3-4  voliunes  of  95  per  cent 
alcohol.  Allow  the  glycogen  precipitate  to  settle,  decant  the  supernatant  fluid, 
and  filter  the  remainder.  Heat  the  glycogen  on  a  water-bath  to  remove  the 
alcohol,  then  subject  it  to  the  following  tests : 

(a)  SolubiUty.— Try  its  solubiUty  in  water  and  10  per  cent  sodium  chloride 
solution. 

(b)  Iodine  Test. — Place  a  small  amount  of  the  glycogen  in  a  depression  of  a 
test-tablet  and  add  2-3  drops  of  dilute  iodine  solution  and  a  trace  of  a  sodiimi 
chloride  solution.  The  same  wine-red  color  is  observed  as  in  the  iodine  test 
upon  the  glycogen  solution. 

3.  Testing  for  Inorganic  Constituents. — (a)  Examination  of  Ash  of  Muscle.— 
Incinerate  a  small  amount  of  muscular  tissue,  dissolve  the  ash  in  dilute  hydro- 
chloric acid.    Test  for  potassium,  phosphates,  magnesium,  calcium  and  chlorides. 

(b)  Demonstration  of  Phosphates  and  Magnesium  in  Muscle  (Hiirthle's 
Experiment). — Tease  a  very  small  piece  of  frog's  muscle  on  a  microscopical 
sUde.  Expose  the  sUde  to  ammonia  vapor  for  a  few  moments,  then  adjust  a 
cover -glass,  and  examine  the  muscle  fibers  under  the  microscope.  Note  the 
large  number  of  crystals  of  ammonium  magnesium  phosphate,  distributed  every- 

NH4-O 

\ 
Mg-0-P=0 

\/ 
O 

where  throughout  the  muscle  fiber,  thus  demonstrating  the  abundance  of  phos- 
phates and  magnesium  in  the  muscle  (Fig.  129,  page  408). 


350  PHYSIOLOGICAL    CHEMISTRY 

Separation  of  Extractives  from  Muscles 

1.  Creatine. — Dissolve  about  lo  grams  of  a  commercial  extract  of  meat  in 
200  c.c.  of  warm  water  (Test  for  Protein  by  Biuret  and  Coagulation  Tests,  see 
Chapter  V.)  Precipitate  the  inorganic  constituents  by  neutral  lead  acetate, 
being  careful  not  to  add  an  excess  of  the  reagent.  "Write  the  equations  for  the 
reactions  taking  place  here.  Allow  the  precipitate  to  settle,  then  filter  and  remove 
the  excess  of  lead  in  the  warm  filtrate  by  hydrogen  sulphide.  Filter  while  the  solu- 
tion is  yet  warm,  evaporate  the  clear  filtrate  to  a  syrup,  and  allow  it  to  stand  at 
least  48  hours  in  a  cool  place.  Crystals  of  creatine  should  form  at  this  point. 
Examine  under  the  microscope  (Fig.  no,  page  342).  Treat  the  syrup  with  200 
c.c.  of  88  per  cent  alcohol,  stir  well  with  a  glass  rod  to  bring  all  soluble  material 
into  solution,  and  then  filter.  The  purine  bases  have  been  dissolved  and  are  in 
the  filtrate,  whereas  the  creatine  crystals  were  insoluble  in  the  88  per  cent  alcohol 
and  remain  on  the  filter  paper.  Wash  the  crystals  with  88  per  cent  alcohol, 
then  remove  them  and  bring  them  into  solution  in  a  little  hot  water.  Decolorize 
the  solution  by  animal  charcoal  and  concentrate  it  to  a  small  volume.  Allow 
the  solution  to  cool  and  note  the  separation  of  colorless  crystals  of  creatine.  ^ 

Make  the  following  tests  on  the  crystals : 

(a)  Microscopical  Examination. — Examine  some  crystals  under  the  micro- 
scope and  compare  the  form  with  those  reproduced  in  Fig.  no,  page  342. 

(b)  Transformation  of  Creatine  into  Creatinine. — Dissolve  the  crystals  in 
about  30  c.c.  of  hot  water.  To  one-half  of  the  solution  in  a  flask  add  an  equal 
volimie  of  normal  hydrochloric  acid  and  heat  on  a  boiUng  water-bath  for  five 
hours  with  reflux  condenser.  The  creatine  has  been  changed  into  creatinine. 
Apply  tests  for  creatinine  as  given  in  Chapter  XXII  to  the  original  solution  as 
well  as  to  the  acidified  solution. 

Diacetyl  Reaction. — To  5  c.c.  of  a  dilute  creatine  solution  add  an  equal  vol- 
ume of  saturated  sodiimi  carbonate  solution  and  a  few  drops  of  a  solution  of 
diacetyl.  A  pink  color  should  develop.  This  test  has  been  made  the  basis  of  a 
method  for  the  quantitative  determination  of  creatine.  ^ 

2.  Hypoxanthine. — Evaporate  the  alcoholic  filtrate  from  the  creatine  to 
remove  the  alcohol.  Make  the  solution  ammoniacal  and  add  ammoniacal  silver 
nitrate  until  precipitation  ceases.  The  precipitate  consists  principally  of  hjrpo- 
xanthine  silver  and  xanthine  silver.  Collect  these  silver  salts  on  a  filter  paper 
and  wash  them  with  water.  Place  the  precipitate  and  paper  in  an  evaporating 
dish  and  boil  for  one  minute  with  nitric  acid  having  a  specific  gravity  of  i.i. 
Filter  while  hot  through  a  double  paper,  wash  with  the  same  strength  of  nitric 
acid  and  allow  the  solution  to  cool.  By  this  treatment  with  nitric  acid  hypo- 
xanthine silver  nitrate  and  xanthine  silver  nitrate  have  been  formed.  The 
former  is  insoluble  in  the  cold  solution  and  separates  on  standing.  After  standing 
several  hours  filter  off  the  hypoxanthine  silver  nitrate  and  wash  with  water  until 
the  wash  water  is  only  slightly  acid  in  reaction.  Examine  the  crystals  of  hypo- 
xanthine silver  nitrate  under  the  microscope  and  compare  them  with  those' in 
Fig.  84,  above.  Now  wash  the  crystals  from  the  paper  into  a  beaker  with  a  Uttle 
water  and  warm  the  liquid.     Remove  the  silver  by  hydrogen  sulphide  and  filter. 

^  For  an  improved  method  of  preparing  pure  creatine  from  creatinine  see  chapter  on 
Physiological  Constituents  of  the  Urine. 
^  Walpole:  Jour.  Physiol.,  42,  301,  1911. 


MUSCULAR   TISSUE 


351 


By  this  means  hypoxanthine  nitrate  has  been  formed  and  is  present  in  the  filtrate. 
(For  crystalUne  form  of  hypoxanthine  nitrate  see  Fig.  40,  p.  136.)  Concentrate 
on  a  water-bath  to  drive  ofif  hydrogen  sulphide,  and  render  the  solution  sUghtly 
alkaUne  with  ammonia.     Warm  for  a  time,  to  remove  the  free  ammonia,  filter. 


Fig.  112. — Hypoxanthine  Silver  Nitrate. 
(Drawn  from  a  student  preparation  by  Dr.  E.  F.  Hirsch.) 

concentrate  the  filtrate  to  a  small  volume  and  allow  it  to  stand  in  a  cool  place. 
Hypoxanthine  should  crystaUize  in  small  colorless  needles.  Examine  the 
crystals  under  the  microscope. 

3.  Xanthine. — To  the  filtrate  from  the  above   experiment  containing  the 


Fig.  113. — Xanthine  Silver  Nitrate. 

xanthine  silver  nitrate  add  ammonia  in  excess.  (The  crystalhne  form  of  xan- 
thine silver  nitrate  is  shown  in  Fig.  113.)  A  brownish -red  precipitate  of 
xanthine  silver  forms.  Treat  this  suspended  precipitate  with  hydrogen  sulphide 
(do  not  use  an  excess  of  hydrogen  sulphide),  warm  the  mixture  for  a  few  mo- 


o^2  PHYSIOLOGICAL  CHEMISTRY 

ments  and  filter  whUe  hot.  Concentrate  the  filtrate  to  a  small  volume  and  put 
away  in  a  cool  place  for  crystallization  (Fig.  in,  page  344).  To  obtam  xanthine 
in  crystalline  form  special  precautions  are  generaUy  necessary.  Evaporate  the 
solution  to  dryness  and  test  according  to  directions  given  in  Chapter  VI  on 
Nucleic  Acids. 


CHAPTER  XX 
NERVOUS  TISSUE 

In  common  with  the  other  sohd  tissues  of  the  body,  nervous  tissue 
contains  a  large  amount  of  water.  The  percentage  of  water  present 
depends  upon  the  particular  form  of  nervous  tissue  but  in  all  forms  it  is 
invariably  greater  in  the  gray  matter  than  in  the  white.  Embryonic 
nervous  tissues  also  contain  a  larger  percentage  of  water  than  the  tissues 
of  adult  life.  The  gray  matter  of  the  brain  of  the  foetus,  for  instance, 
contains  about  92  per  cent  of  water,  whereas  the  gray  matter  of  the 
brain  of  the  adult  contains  but  83-84  per  cent  of  the  fluid. 

Among  the  solid  constituents  of  nervous  tissue  are  proteins,  choles- 
terol, cerchrosides  (cerebrin,  etc.),  lecithin,  kephalin,  protagon  {?),  para- 
nucleoprotagon,  miclein,  neurokeratin,  collagen,  extractives,  and  inorganic 
salts.  The  proteins  are  present  in  the  greatest  amount  and  comprise 
about  50  per  cent  of  the  total  solids.  Three  distinct  proteins,  two 
globulins,  and  a  nucleoprotein,  have  been  isolated  from  the  nervous 
tissue.  The  globulins  coagulate  at  47°C.  and  7o-75°C.,  respectively, 
while  the  nucleoprotein  coagulates  at  56-6o°C.  This  nucleoprotein 
contains  about  0.5  per  cent  of  phosphorus  (Halliburton,  Levene.) 
Nervous  tissue  is  composed  of  a  relatively  large  quantity  of  a  variety 
of  compounds  which  collectively  may  be  grouped  under  the  term 
"lipoid" — substances  resembling  the  fats  in  some  of  their  physical 
properties  and  reactions  but  distinct  in  their  composition.  We  will 
class  cholesterol,  the  cerebrosides  and  the  phosphorized  fats  as  lipoids. 

The  consideration  of  lipoids  (or  lipins'^)  is  assuming  added  impor- 
tance. These  substances  constitute  one  of  the  two  great  groups  of 
tissue  colloids,  the  proteins  being  the  remaining  group.  So  far  as  struc- 
ture and  chemical  properties  are  concerned  the  various  classes  of  lipoids 
are  entirely  unlike. 

The  group  of  phosphorized  fats  are  very  important  constituents  of 
nervous  tissue.  The  best  known  members  of  this  group  are  IccitJiin 
prolagon  (?)  and  kephalin.  Lecithin  occurs  in  larger  amount  than 
the  other  members  of  the  group,  has  been  more  thoroughly  studied 
than  the  others  and  is  apparently  of  greater  importance.  Upon  de- 
composition lecithin  yields  fatty   acids,   glycero- phos phoric  acid,   and 

'  Rosenbloom  and  Gics:  Biochemical  Bulletin,  i,  51,  191 1.  The  term  lipoid  was  intro- 
duced by  Overton  (Studien  iiber  die  Narkose,  Jena,  1901,  Gustav  Fischer). 

23  353 


354  PHYSIOLOGICAL   CHEMISTRY 

choline.     Each   lecithin   molecule    contains    two    fatty   acid   radicals 

which  may  be  those  of  the  same  or  different  fatty  acids.     Thus  we  have 

different  lecithins  depending  upon  the  particular  fatty  acids  radicals 

which  are  present  in  the  molecule.     The  formula  of  a  typical  lecithin 

would  be  the  following. 

CHo— OOC.C17H30 

I 
CH— OOC.C17H35 

I 
CH2O— PO— O.C2H4 

\ 
(CH3)3  =  N 

/ 
OH  HO 

This  lecithin  would  be  called  distearyl-lecithin  or  choline-distearyl- 
glycero-phosphoric  acid.  Upon  decomposition  the  molecule  splits  ac- 
cording to  the  following  reaction: 

C44H90NPO9  +  3H2O  -^  2C18H36O2  +  C3H9PO6  +  C6H15NO2. 

Lecithin.  Stearic  acid.        Glycero-phosphoric  acid.  Choline. 

The  lecithins  are  not  confined  to  the  nervous  tissues  but  are  found  in 
nearly  all  animal  and  vegetable  tissues.  Lecithin  is  a  primary  con- 
stituent of  the  cell.  It  is  soluble  in  chloroform,  ether,  alcohol,  benzene, 
and  carbon  disulphide.  The  chloroform  or  alcohol-ether  solution 
may  be  precipitated  by  acetone.  Lecithin  may  be  caused  to  crystal- 
lize in  the  form  of  small  plates  by  cooling  the  alcoholic  solution  to  a 
low  temperature.  It  has  the  power  of  combining  with  acids  and  bases, 
and  the  hydrochloric  acid  combination  has  the  power  of  forming  a 
double  salt  with  platinic  chloride. 

Choline,  as  was  indicated  above,  is  one  of  the  decomposition 
products  of  lecithin.  It  is  trimethyl-hydroxyethyl-ammonium  hydroxide 
and  has  the  following  formula: 

CHz.CHaCOH) 

/ 

N^(CH3)3 

\ 
OH 

Researches  have  shown  that  great  importance  is  to  be  attached  to  the 
detection  of  choline  in  the  cerebro-spinal  fluid  and  the  blood  in  certain 
cases  of  degenerative  disease  of  the  nervous  system.  In  this  connec- 
tion tests  for  choline  (see  page  357)  are  of  interest  and  value. 

Protagon,  another  nitrogenous  phosphorized  substance,  is  a  body 
over  which  there  has  been  much  discussion.     Upon  decomposition  it 


NERVOUS   TISSUE  355 

is  said  by  some  investigators  to  yield  cerebrin  and  the  decomposition 
products  of  lecithin.  It  has  been  shown  by  Posner  and  Gies^  as  well 
as  by  Rosenheim  and  Tebb-  that  protagon  is  a  mixture  and  has  no 
existence  as  a  chemical  individual.  Koch'  reported  data  obtained 
from  purified  preparations  which  indicate  that  protagon  contains  at 
least  three  substances:  "a  phosphatide  containing  cholin,  a  cerebro- 
side  containing  sugar,  a  complex  combination  of  a  cholin-free  phos- 
phatide with  a  cerebroside  to  which  an  ethereal  sulphuric  acid  group 
is  attached."  On  the  basis  of  his  data,  he  believed  the  term  pro- 
tagon to  have  no  chemical  significance.  He  proposed  the  term  sul- 
phatide.     Koch's  preparation  analyzed  as  follows  (per  cent) : 

Choline  Sugar  Nitrogen       Phosphorus        Sulphur 

i.o  12. o  2,3  1.7  1.9 

He  suggested  the  following  structure: 

O 

11 

Phosphatide^ — O — S — O — Cerebroside 

grouping  |,  grouping 

O 

Kephalin  is  the  third  member  of  the  group  of  phosphorLzed  fats. 
It  is  precipitated  from  its  acetone-ether  extract  by  alcohol.  It  contains 
about  4  per  cent  of  phosphorus  and  has  been  given  the  formula  C42H79- 
NPO13.     Kephalin  may  be  a  stage  in  lecithin  metabolism. 

The  cerebrosides  are  substances  containing  nitrogen  but  no  phos- 
phorus, and  are  important  constituents  of  the  white  matter  of  nervous 
tissue.  Certain  ones  have  also  been  found  in  the  spleen,  pus,  and  in  egg 
yolk.  They  may  be  extracted  from  the  tissue  by  boiling  alcohol  and  are 
insoluble  in  cold  alcohol,  cold  and  hot  ether,  and  in  water  and  dilute 
alkalis.  The  cerebroside  termed  cerebrin  is  a  mixture  containing  phre- 
nosin  (pseudo-cerebrin  or  cerebron),  a  body  yielding  the  carbohydrate 
galactose  on  decomposition. 

Cholesterol,  one  of  the  primary  cell  constituents,  is  present  in  fairly 
large  amount  in  nervous  tissue.  It  occurs  in  two  forms,  i.e.,  free  and 
combined  as  an  ester.  It  is  claimed*  that  99  per  cent  of  the  choles- 
terol of  brain  tissue  (boy)  is  in  the  free  state.  It  is  a  mon-atomic 
alcohol  containing  at  least  one  double  bond  and  possesses  the  formula 
C27H46OH  or  C27H43OH.  There  is  still  some  uncertainty  as  to  the 
exact  structure  of  cholesterol.     It  may  possess  a  terpene  structure.    It 

^  Posner  and  Gies:  Journal  of  Biological  Chemistry,  i,  59,  1905-06. 
^  Rosenheim  and  Tebb:  Journal  of  Physiology,  36  and  37,  1907-8. 
'  Koch:  Journal  Biological  Chemisly,  ii,  March,  191  2,  Proceedings. 
*Lapworth:  Jour.  Path.  Bad.,  1911,  p   256. 


356  PHYSIOLOGICAL    CHEMISTRY 

was  formerly  called  a  " non-saponifiable  fat"  but  since  it  is  not  changed 
in  any  way  by  boiling  alkalis  it  is  not  a  fat.  It  is  soluble  in  ether, 
chloroform,  benzene,  and  hot  alcohol.  It  crystallizes  in  the  form  of 
thin,  colorless,  transparent  plates  (Fig.  57,  page  210).  Cholesterol 
is  present  in  bile,  occurs  abundantly  in  one  form  of  biliary  calculus. 
It  is  also  present  in  blood  and  its  quantitative  determination  is  of 
clinical  importance  (see  Chapter  XVI).  It  has  been  found  in  feces, 
wool  fat,  egg  yolk,  and  milk,  frequently  in  the  form  of  its  esters  of 
higher  fatty  acids.  It  is  generally  believed  that  the  cholesterol  present 
in  the  animal  body  has  its  origin  in  the  vegetable  kingdom.  Some 
evidence  has  been  submitted^  indicating  a  synthesis  of  cholesterol 
under  certain  conditions  in  the  animal  body.  However,  it  is  probable 
that  cholesterol  is  not  readily  synthesized  in  the  body.- 

Paranucleoprotagon  is  a  phosphorized  substance  originally  isolated 
from  brain  tissue  by  Ulpiani  and  Lelli  and  recently  reinvestigated  by 
Steel  and  Gies.     It  is  said  to  possess  lecithoprotein  characteristics. 

Nervous  tissue  yields  about  i  per  cent  of  ash  which  is  made  up  in 
great  part  of  alkaline  phosphates  and  chlorides. 

Experiments  on  the  Lipoids  of  Nervous  Tissue^ 

1.  Preparation  of  Lecithin.^ — Treat  the  finely  divided  brain  of  a  sheep  with 
ether  and  allow  it  to  stand  in  the  cold  for  48-72  hours.  The  cold  ether  will  ex- 
tract lecithin  and  cholesterol.  Filter  and  add  acetone  to  the  filtrate  to  pre- 
cipitate the  lecithin.     Filter  off  of  the  lecithin  and  test  it  as  follows : 

(a)  Microscopical  Examination. — Suspend  a  small  portion  in  a  drop  of 
water  on  a  sUde  and  examine  under  the  microscope. 

fb)  Osmic  Acid  Test.^ — Treat  a  small  portion  with  osmic  acid.  What 
happens? 

(c)  Acrolein  Test. — Make  the  acrolein  test  according  to  directions  on  page 
180. 

(d)  Test  for  Phosphorus. — See  page  129,  Chapter  VI. 

2.  Preparation  of  Cholesterol. — Place  a  small  amount  of  finely  divided  brain 
tissue  under  ether  and  stir  occasionally  for  one  hour.  Filter,  evaporate  the 
filtrate  to  dryness  on  a  water-bath,  and  test  the  cholesterol  according  to  direc- 
tions given  below.     (If  it  is  desired,  the  ether  extract  from  the  so-called  protagon, 

1  Klein:  Biochem.  Zeit.,  30,  465,  1910. 

2  Gardner  and  Lander:  Proc.  Royal  Soc,  London  (B),  87,  229,  1913. 
^Preparation  of  So-called  Protagon. — Divide  the  brain  of  a  sheep  into  small  pieces, 

treat  with  85  per  cent  alcohol  and  warm  on  a  water-bath  45°C.  for  two  hours.  Filter  hot 
into  a  bottle  or  strong  flask  and  cool  to  o°C.  for  one-half  hour  by  means  of  a  freezing  mix- 
ture. By  this  procedure  both  protagon  and  cholesterol  are  caused  to  precipitate.  Filter 
the  cold  solution  rapidly  and  treat  the  precipitate  on  the  paper  with  ice  cold  ether  to  dis- 
solve out  the  cholesterol.  The  protagon  may  now  be  redissolved  in  warm  85  per  cent 
alcohol  from  which  solution  it  will  precipitate  upon  cooling. 

*  For  the  preparation  of  lecithin  in  purer  form  see  MacLeon:  Jour.  Path.  Bad.,  18,  490, 

^  Osmic  acid  serves  to  detect  fats  which  contain  unsaturated  fatty  acid  radicals,  e.g., 
oleic  acid,  in  their  molecule. 


NERVOUS   TISSUE 


357 


or  the  ether-acetone  filtrate  from  the  lecithin  may  be  used  for  the  isolation  of 
cholesterol.  In  these  cases  it  is  simply  necessary  to  evaporate  the  solution  to 
dryness  on  a  water-bath. )  Upon  the  cholesterol  prepared  by  either  of  the  above 
methods  make  the  following  tests  : 

((7)  Microscopical  Examination. — Examine  the  crystals  under  the  microscope 
and  compare  them  with  those  in  Fig.  57,  page  210. 

(b)  HjSOi  Test  (Salkowskii. — Dissolve  a  few  crystals  of  cholesterol  in  a 
little  chloroform  and  add  an  equal  volume  of  concentrated  sulphuric  acid. 
A  play  of  colors  from  bluish-red  to  cherry-red  and  purple  is  noted  in  the  chloro- 
from,  while  the  acid  assumes  a  marked  green  fluorescence. 

(c)  Acetic  Anhydride-  Hi^SO^  Test  (Liebermann-Burchard*. — Dissolve  a  few 
crystals  of  cholesterol  in  2  c.c.  of  chloroform  in  a  dry  test-tube.  Now  add  10 
drops  of  acetic  anhydride  and  1-3  drops  of  concentrated  ^Iphuric  acid.  The 
solution  becomes  red,  then  blue,  and  finally  bluish-green  in  color. 

(d)  Iodine-sulphuric  Acid  Test. — Place  a  few  crystals  of  cholesterol  in  one  of 
the  depressions  of  a  test-tablet  and  treat  with  a  drop  of  concentrated  sulphuric  acid 
and  a  drop  of  a  very  dilute  solution  of  iodine.  A  play  of  colors,  consisting  of  violet, 
blue,  green,  and  red,  results. 

(e)  Schif's  Reaction. — To  a  little  cholesterol  in  an  evaporating  dish  add  a  few 
drops  of  a  reagent  made  by  adding  i  volume  of  10  per  cent  ferric  chloride  to  3  vol- 
umes of  concentrated  sulphuric  acid.  Evaporate  to  drj'ness  over  a  low  flame  and 
observe  the  reddish-violet  residue  which  changes  to  a  bluish- violet. 

(/)  Phosphorus. — Test  for  phosphorus  according  to  directions  given  in  Chapter 
VI,  page  129.     Is  phosphorus  present? 

3.  Preparation  of  Cerebrin.  —Treat  100  grams  of  finely  divided  brain  tissue, 
in  a  flask,  with  200  c.c.  of  95  per  cent  alcohol  and  boil  on  a  water-bath  for  one- 
half  hour,  keeping  the  volume  constant  by  adding  fresh  alcohol  as  needed  or  by 
the  use  of  a  reflux  condenser.  Filter  the  solution  hot  and  stand  the  cloudy 
filtrate  away  for  24  hours.  (If  the  filtrate  is  not  cloudy  concentrate  it  upon  the 
water-bath  until  it  is  so.j  Filter  off  the  cerebrin  (cerebrin,  lecithin,  kephaUn, 
cholesterol  1  and  test  it  as  follows : 

fa)  Microscopical  Examination. — Suspend  a  small  portion  in  a  drop  of  water 
on  a  slide  and  examine  under  the  microscope. 

(bi  SolubiUty. — Try  the  solubiUty  of  cerebrin  in  water,  10  per  cent  sodium 
chloride  and  in  dilute  acid  and  alkaU,  and  in  hot  and  cold  alcohol  and  hot  and 
cold  ether. 

(c)  Phosphorus. — Test  for  phosphorus  according  to  directions  in  Chapter 
VI,  page  129.     How  does  the  result  compare  with  that  on  lecithin? 

(dj  Place  a  Uttle  cerebrin  on  platinum  foil  and  warm.     Note  the  odor. 

(e)  Hydrolysis  of  Cerebrin. — Place  the  remaining  cerebrin  in  a  small  evaporat- 
ing dish,  add  equal  volumes  of  water  and  dilute  hydrochloric  acid,  and  boil  for 
one  hour.  Cool,  neutralize  with  sohd  potassium  hydroxide,  filter,  and  test 
with  Fehling's  solution.  Is  there  any  reduction,  and  if  so  how  do  you  explain 
it? 

4.  Tests  for  ChoUne.  (a)  Rosenheim's  Per  iodide  rt'^/.— Prepare  an  alcoholic 
extract  of  the  fluid  under  examination,  and  after  evaporation  apply  Rosenheim's 
iodo-potassium  iodide  solution'  to  a  little  of  the  residue.     In  a  short  time  dark 

'  Prepared  by  dissolving  2  grams  of  iodine  and  6  grams  of  potassium  iodide  in  100  cc. 

water. 


358  PHYSIOLOGICAL   CHEMISTRY 

brown  plates  and  prisms  of  choline  periodide  begin  to  form  and  may  be  detected  by 
means  of  the  microscope.  Occasionally  they  are  large  enough  to  be  visible  to  the 
naked  eye.  They  somewhat  resemble  crystals  of  hemin  (see  page  265).  If  the 
slide  be  permitted  to  stand,  thus  allowing  the  fluid  to  evaporate,  the  crj'stals  will 
disappear  and  leave  brown  oUy  drops.  They  will  reappear,  however,  upon  the 
addition  of  fresh  iodine  solution,  v.  Stanek  claims  that  this  choline  compound 
has  the  formula  CsHnNOI.Ig. 

{h)  Rosenheim' s  Bismuth  Test. — Extract  the  fluid  under  examination  with 
absolute  alcohol,  evaporate,  and  reextract  the  residue.  Repeat  the  extraction 
several  times.  Dissolve  the  final  residue  in  2-3  c.c.  of  water  and  add  a  drop  of 
Kraut's  reagent.^  Choline  is  indicated  by  the  appearance  of  a  bright  brick-red 
precipitate. 

^  Dissolve  272  grams  of  potassium  iodide  in  water  and  add  80  grams  of  bismuth  sub- 
nitrate  dissolved  in  200  grams  of  nitric  acid  (sp.  gr.  1.18).  Permit  the  potassium  nitrate  to 
crystallize  out,  then  filter  it  off  and  make  the  filtrate  up  to  i  liter  with  water. 


CHAPTER  XXI 

URINE:    GENERAL    CHARACTERISTICS    OF    NORMAL    AND 
PATHOLOGICAL  URINE 

Volume. — The  volume  of  urine  excreted  by  normal  indiv-iduals 
during  any  definite  period  fluctuates  within  very  wide  limits.  The 
average  output  for  twenty-four  hours  is  placed  by  German  writers 
between  1500  and  2000  c.c.  This  value  is  not  strictly  applicable  to  con- 
ditions in  America,  however,  since  it  has  been  found  that  the  average 
normal  excretion  of  the  adult  male  American  falls  within  the  lower 
values  of  1000-1200  c.c.  The  volume-excretion  is  influenced  greatly 
by  the  diet,  particularly  by  the  ingestion  of  fluids. 

Certain  pathological  conditions  cause  the  output  of  urine  for  any 
definite  period  to  depart  very  decidedly  from  the  normal  output. 
Among  the  pathological  conditions  in  which  the  volume  of  urine  is  i«- 
creased  above  normal  are  the  following:  Diabetes  mellitus,  diabetes 
insipidus,  certain  diseases  of  the  nervous  system,  contracted  kidney, 
amyloid  degeneration  of  the  kidney,  and  in  convalescence  from  acute 
diseases  in  general.  Many  drugs  such  as  calomel,  digitalis,  acetates, 
and  salicylates  also  increase  the  volume  of  the  urine  excreted.  A 
decrease  from  the  normal  is  observed  in  the  following  pathological 
conditions:  Acute  nephritis,  diseases  of  the  heart  and  lungs,  fevers, 
diarrhoea,  and  vomiting. 

Color. — Normal  urine  ordinarily  possesses  a  yellow  tint,  the  depth 
of  the  color  being  dependent  in  part  upon  the  density  of  the  fluid.  The 
color  of  normal  urine  is  due  principally  to  a  pigment  called  nrochrome:^ 
traces  of  hemato porphyrin,  urobilin,  and  uroerythrin  have  also  been 
detected.  Under  pathological  conditions  the  urine  is  subject  to  pro- 
nounced variations  in  color  and  may  contain  many  varieties  of  pig- 
ments. Under  such  circumstances  the  urine  may  vary  in  color  from  an 
extremely  light  yellow  to  a  verj^  dark  brown  or  black.  Vogel  has  con- 
structed a  color  chart  which  is  of  some  value  for  purposes  of  comparison. 
The  nature  and  origin  of  the  chief  variations  in  the  urinary  color  are  set 
forth  in  tabular  form  by  Halliburton  as  follows: 

^  Urochrome  is  believed  to  be  identical  with  the  yellow  pigment  (lactochrome)  of  milk 
whey  (Palmer  and  Coolidge:  Jour.  Biol.  C/tem.,  17,  251,  1914). 

359 


360  PHYSIOLOGICAL   CHEMISTRY 


Color 

Cause  of  coloration 

Pathological  condition 

Nearly  colorless 

Dilution,   or  diminution  of 
normal  pigments. 

'  Nervous  conditions:  hy- 
druria,  diabetes  insipidus, 
granular  kidney. 

Dark  j^ellow  to  brown- red.  . 

Increase  of  normal,  or  oc- 
currence   of     pathological, 
pigments.        Concentrated 
urine. 

Acute  febrile  diseases. 

MUky 

Fat  globules 

Chyluria. 

Pus  corpuscles 

Purulent  diseases  of  the 
urinary  tract. 

Orange  

Excreted  drugs 

Santonin,  crysophanic  acid. 

Red  or  reddish 

Hematoporphyrin 

Unchanged  hemoglobin 

Hemorrhages,  or  hemoglo- 
binuria,. 

Pigments  in  food  (logwood, 
madder,  bilberries,  fuchsin). 

Brown  to  brown  black 

Hematin 

Small  hemorrhages. 

Methemoglobin 

Methemoglobinuria. 

Melanin 

Melanotic  sarcoma. 

Hydrochinol  and  catechol  .  . 

Carbolic-acid  poisoning. 

Greenish    yellow,    greenish 
brown,  approaching  black.. 

Bile-pigments 

Jaundice. 

1 
Dirty  green^  or  blue 

A  dark  blue  scum  on  surface, 
with  a  blue  deposit,  due  to 
an  excess  of  indigo-forming 
substances. 

Cholera,  typhus;  seen  espe- 
cially when  the  urine  is 
putrefying. 

Brown-yellow  to  red-brown, 
becoming    blood-red    upon 
adding  alkalis. 

Substances      contained      in 
senna,  rhubarb    and  cheli- 
donium    which    are    intro- 
duced into  the  system. 

Transparency. — Normal  urine  is  ordinarily  perfectly  clear  and 
transparent  when  voided.  On  standing  for  a  variable  time,  however,  a 
cloud  (nubecula)  consisting  principally  of  nucleoprotein  or  mucoid  (see 
page  396)  and  epithelial  cells  forms.  A  turbidity  due  to  the  precipita- 
tion of  phosphates  is  normally  noted  in  urine  passed  after  a  hearty 

1  This   dirty  green  or  blue  color  also  occurs  after  the  use  of  methylene  blue  in  the 
organism. 


URINE  361 

meal.  The  urine  obtained  2-3  hours  after  a  meal  or  later  is  ordinarily 
free  from  turbidity.  Permanently  turbid  urines  ordinarily  arise  from 
pathological  conditions. 

Odor. — The  odor  of  normal  urine  is  of  a  faint,  aromatic  type.  The 
bodies  to  which  this  odor  is  due  are  not  well  known,  but  it  is  claimed  by 
some  investigators  to  be  due,  at  least  in  part,  to  the  presence  of  minute 
amounts  of  certain  volatile  organic  acids.  Dehn  and  Hartman^  have 
recently  succeeded  in  isolating  from  urine  a  neutral  ill-smelling  substance 
which  they  call  urinod.  Its  empirical  formula  is  CeHsO.  Urinod  occurs 
in  urine  to  the  extent  of  only  1-2  parts  in  100,000  parts  of  urine. 
When  the  urine  undergoes  decomposition,  e.g.,  in  alkaline  fermentation, 
a  very  unpleasant  ammoniacal  odor  is  evolved.  All  urines  are  subject 
to  such  decomposition  if  allowed  to  stand  for  a  sufficiently  long  time. 
Under  normal  conditions  the  urine  very  often  possesses  a  peculiar  odor 
due  to  the  ingestion  of  some  certain  drug  or  vegetable.  For  instance, 
cubebs,  copaiba,  myrtol,  saffron,  tolu,  and  turpentine  each  imparts  a 
somewhat  specific  odor  to  the  urine.  After  the  ingestion  of  asparagus, 
the  urine  also  possesses  a  typical  odor  due  to  the  formation  of  methyl 
mercaptan  (CH3  SH)  in  the  intestine. 

Frequency  of  Urination. — 'The  frequency  of  urination  varies  greatly 
in  different  individuals,  but  in  general  is  dependent  upon  the  amount  of 
fluid  in  the  bladder.  In  pathological  conditions  an  inflammatory  affec- 
tion of  the  urinary  tract  or  any  disturbance  of  the  innervation  of  the 
bladder  will  influence  the  frequency.  Affections  of  the  spinal  cord 
which  lead  to  an  increased  irritability  of  the  bladder  or  a  weakening  of 
the  sphincter,  or  any  condition  lowering  the  residual  capacity  of  the 
bladder,  will  result  in  increasing  the  frequency  of  urination. 

Reaction. — The  mixed  24-hour  urinary  excretion  ot  a  normal  indi- 
vidual ordinarily  possesses  an  acid  reaction  to  litmus.  This  acidity  in 
normal  cases  is  represented  on  the  average  by  a  hydrogen  ion  concentra- 
tion of  loXio"^,  although  it  may  vary  from  0.40  to  150X10"'.  The 
reaction  of  the  urine  represents  an  equilibrium  between  a  large  number 
of  acid  and  basic  constituents,  both  organic  and  inorganic,  which  it  con- 
tains. Organic  acids  and  bases  play  a  part  in  producing  the  normal 
reaction,  but  this  is  probably,  in  the  main,  dependent  upon  the  relative 
amounts  of  the  mono-  and  dibasic  sodium  and  potassium  phosphates. 
The  monobasic  sodium  phosphate  (XaH>P04)  is  acid  in  reaction,  while 
the  dibasic  phosphate  (Na2HP04)  is  alkaline  in  reaction.  The  ex- 
cretion of  acid  or  alkaline  phosphate  by  the  kidneys  is  one  of  the  factors 
in  the  regulation  of  the  neutrality  of  the  blood  and  of  the  organism 
in  general.     The  acidity  of  the  urine  as  determined  by  titration  runs 

'Dehn  and  Hartman:  Jour.  .\m.  Chan.  Soc,  36,  2136    1914. 


362 


PHYSIOLOGICAL   CHEMISTRY 


in  general  parallel  with  the  hydrogen  ion  concentration  and  seems  to  be 
dependent  upon  the  same  factors,  and  in  more  acid  urines  mainly  on 
the  phosphate  content.  (For  further  discussion  of  acidity  see  Chapter 
VIII  on  Gastric  Analysis.) 

The  mean  acidity  in  cardio-renal  diseases  is  high — about  50X10" '^ 
as  compared  with  loXio"^,  the  normal  mean.  In  general  the  acidity 
tends  to  be  increased  in  the  greater  number  of  pathological  disorders. 

The  composition  of  the  food  is  perhaps  the  most  important  factor 
in  determining  the  reaction  of  the  urine  (see  Chapter  XXVII  on  Met- 
abolism for  influence  of  base-forming  and  acid-forming  foods).  The 
reaction  ordinarily  varies  considerably  according  to  the  time  of  day 
the  urine  is  passed.  For  instance,  for  a  variable  length  of  time  after 
a  meal  the  urine  may  be  neutral  or  even  alkaline  in  reaction  to  litmus, 


/o:-.',-!^'..'!  Tj''-U  '-2.' 


Fig.  114. — Deposit  in  Ammoniacal  Fermentation. 
a,  Acid  ammonium  urate;  b,  ammonium  magnesium  phosphate;  c,  bacteria. 

owing  to  the  claim  of  the  gastric  juice  upon  the  acidic  radicals  to  further 
the  formation  of  hydrochloric  acid  for  use  in  carrying  out  the  digestive 
secretory  function.  This  change  in  reaction  is  known  as  the  alkaline 
tide  and  is  common  to  perfectly  healthy  individuals.  The  urine  may 
also  become  temporarily  alkaline  in  reaction  to  litmus,  as  the  result  of 
ingesting  alkaline  carbonates  or  certain  salts  of  tartaric  and  citric  acids 
which  may  be  transformed  into  carbonates  within  the  organism. 
Normal  urine  upon  standing  for  some  time  becomes  alkaline  in  reaction 
to  litmus,  owing  to  the  inception  of  alkaline  or  ammoniacal  fermentation 
through  the  agency  of  micro-organisms.  This  fermentation  has  no 
especial  diagnostic  value  except  in  cases  where  the  urine  has  undergone 
this  change  within  the  organism  and  is  voided  in  the  decomposed  state. 
Ammoniacal  fermentation  is  ordinarily  due  to  cystitis  or  occurs  as  the 
result  of  infection  in  the  process  of  catheterization.     A  microscopical 


URINE  363 

examination  of  such  urine  (Fig.  114)  shows  the  presence  oi  ammonium 
magnesium  phosphate  crystals,  amorphous  phosphates,  and  not  infre- 
quently afumonium  urate. 

Ingestion  of  acid  fruits  (oranges,  lemons,  peaches,  etc.)  causes 
the  formation  of  alkaline  urine.  This  is  due  to  the  fact  that  the  ash  of 
such  fruits  is  alkaline  and  when  the  fruits  are  combusted  in  the  body 
carbonates  are  formed.  On  the  other  hand,  bread,  cereals,  etc.,  yield  an 
acid  ash  and  an  acid  urine. 

Occasionally  a  urine  which  possesses  a  normal  acidity  when  voided, 
upon  standing  instead  of  undergoing  ammoniacal  fermentation  as  above 
described,  will  become  more  strongly  acid  in  reaction.  Such  a  phe- 
nomenon is  termed  acid  fermentation.  Accompanying  this  increased 
acidity  there  is  ordinarily  a  deepening  of  the  tint  of  the  urinary  color. 


Fig.  115. — Deposit  in   Acid   Fermentation. 
a,  Fungus;  b,  amorphous  sodium  urate;  e,  uric  acid;  d,  calcium  oxalate. 

Such  urines  may  contain  acid  urates,  uric  acid,  fungi,  and  calcium  oxalate 
(Fig.  115,  above).  On  standing  for  a  sufficiently  long  time  any  urine 
which  exhibits  acid  fermentation  will  ultimately  change  in  reaction, 
due  to  the  inception  of  alkaline  fermentation,  and  will  show  the  micro- 
scopical deposits  characteristic  of  such  a  urine. 

Specific  Gravity. — The  specific  gravity  of  the  urine  of  normal  indi- 
viduals varies  ordinarily  between  1.015  and  1.025.  This  value  is  sub- 
ject to  wide  fluctuations  under  various  conditions.  For  instance, 
following  copious  water-  or  beer-drinking  the  specific  gravity  may  fall 
to  1.003  or  lower,  whereas  in  cases  of  excessive  perspiration  it  may  rise 
as  high  as  1.040  or  even  higher.  Where  a  very  accurate  determina- 
tion of  the  specific  gravity  is  desired  use  is  commonly  made  of  the 
Pyknometer  or  of  the  Westphal  hydrostatic  balance.  These  instruments, 
however,  are  not  suited  for  clinical  use.     The  clinical  method  of  deter- 


364 


PHYSIOLOGICAL   CHEMISTRY 


mining  the  specific  gravity  is  by  means  of  a  urinometer  (Fig.  116).  This 
affords  a  very  rapid  method  and  at  the  same  time  is  sufficiently  accurate 
for  clinical  purposes.  The  urinometer  is  always  calibrated  for  use  at  a 
specific  temperature  and  the  observations  made  at  any  other  tem- 
perature must  be  subjected  to  a  certain  correction  to  obtain  the  true 
specific  gravity.  In  making  this  correction  one  unit  of  the  last  order  is 
added  to  the  observed  specific  gravity  for  every  three  degress  above 
the  normal  temperature  and  subtracted  for  every  three 
degrees  below  the  normal  temperature.  For  instance, 
if  in  using  a  urinometer  calibrated  for  i5°C.  the  specific 
gravity  of  a  urine  having  a  temperature  of  2i°C.  is 
determined  as  1.018  it  is  necessary  to  add  to  the 
observed  specific  gravity  two  units  of  the  third  order 
to  obtain  the  real  specific  gravity  of  the  urine.  There- 
fore the  true  specific  gravity,  at  i5°C.,  of  a  urine 
having  a  specific  gravity  of  1.018  at  2i°C.  is  1.018  + 
0.002  =  1.020. 

Pathologically,  the  specific  gravity  may  be  sub- 
jected to  very  wide  variations.  This  is  especially 
true  in  diseases  of  the  kidneys.  In  acute  nephritis 
ordinarily  the  urine  is  concentrated  and  of  a  high 
specific  gravity,  whereas  in  chronic  nephritis  the  re- 
I  If        i  verse  conditions  are  more  apt  to  prevail.     In  fact, 

under  most  conditions,  whether  physiological  or  patho- 
logical, the  specific  gravity  of  the  urine  is  inversely 
proportional  to  the  volume  excreted.  This  is  not 
true  of  diabetes  mellitus,  however,  where  the  volume 
of  urine  is  large  and  the  specific  gravity  is  also  high, 
owing  to  the  sugar  contained  in  the  urine. 
Fig.  116.— Urin-  The  amount  of  solids  eliminated  in  the  excretion 
OMETER  AND  Cylin-  £qj.  twcnty-four  hours  may  be  roughly  calculated  by 
means  of  Long's  coefficient,  i.e.,  2.6.  The  solid  con- 
tent of  1000  c.c.  of  urine  is  obtained  by  multiplying  the  last  two 
figures  of  the  specific  gravity  observed  at  2  5°C.  by  2.6.  To  determine 
the  amount  of  solids  excreted  in  twenty-four  hours  if  the  volume  was 
1 1 20  c.c.  and  the  specific  gravity  was  1.018  the  calculation  would  be  as 
follows : 

{a)   18X2.6  =  46.8  grams  of  solid  matter  in  1000  c.c.  of  urine. 
A6.8X1120 


(&) 


1000 


52.4  grams   of  solid  matter  in  11 20  c.c.  of  urine. 


The  coefficient  of  Haser  (2.33)  which  has  been  in  use  for  years  prob- 
ably gives  values  that  are  inaccurate  for  conditions  existing  in  America. 


URINE 


365 


This  coefficient  was  calculated  on  the  basis  of  the  specific  gravity  deter- 
mined at  a  temperature  of  i5°C. 

Freezing-point  (Cryoscopy). — The  freezing-point  of  a  solution  de- 
pends upon  the  total  number  of  molecules  of  solid  matter  dissolved  in 
it.  The  determination  of  the  osmotic  pressure 
by  this  method  has  come  to  be  of  some  clinical 
importance,  particularly  as  an  aid  in  the  diag- 
nosis of  kidney  disorders.  In  this  connection 
it  is  best  to  collect  the  urine  from  each  kidney 
separately  and  determine  the  freezing-point  in 
the  individual  samples  so  collected.  By  this 
means  considerable  aid  in  the  diagnosis  of  renal 
diseases  may  be  secured.  The  fluids  most  fre- 
quently examined  cryoscopically  are  the  blood 
(see  page  245)  and  the  urine.  The  freezing- 
point  is  denoted  by  A.  The  value  of  A  for 
normal  urine  varies  ordinarily  between  —1.3° 
and  —  2.3°C.,  the  freezing-point  of  pure  water 
being  take  as  0°.  A  is  subject  to  very  wide 
fluctuations  under  unusual  conditions.  For 
instance,  following  copious  water-  or  beer- 
drinking    A    may  have    as    high    a    value    as 

—  o.2°C.,  whereas  on  a  diet  containing  much 
salt  and  deficient  in  fluids  the  value  of  A  may 
be  lowered  to  —  3°C.  or  even  lower.  The  freez- 
ing point  of  normal  blood  is  generally  about 

—  o.56°C.  and  is  not  subject  to  the  wide 
variations  noted  in  the  urine,  because  of  the 
tendency  of  the  organism  to  maintain  the 
normal  osmotic  pressure  of  the  blood  under  all 
conditions.  Variations  between  —0.51°  and 
o.62°C.  may  be  due  entirely  to  dietary  con- 
ditions, but  if  any  marked  variation  is  noted 
it  can,  in  most  cases,  be  traced  to  a  disordered 
kidney  function. 

T-.         •  •    i.  J   i.         •      i.'  1  1       which    the   mixture    to   be 

r  reezmg-pomt  determmations  may  be  made    observed   is  placed.    Two 

by  means  of  the  Beckmann-Heidenhain  appa-    stirrers  are  shown,  one  for 

,  the  coohng  mi.xture  in  the 

ratUS   (Fig.  117)  or  the  Zikel  pektOSCOpe.      The    jar  and  one  for  the  experi- 

Beckmann-Heidenhain    apparatus    consists   of    ^^^^^  mixture, 
the  following  parts:     A  strong  battery  jar  or  beaker   (C)   furnished 
with  a  metal  cover  which  is  provided  with  a  circular  hole  in  its  center. 
This  strong  glass  vessel  serves  to  hold  the  freezing  mixture  by  means 


Fig.  117. — Beckmann- 
Heidenhain  Freezing- 
point  Apparatus.     (Long.) 

D,  a  delicate  thermom- 
eter; C,  the  containing 
jar;  B,  the  outside  or  air 
mantle  tube;  A,  the  tube  in 


366  PHYSIOLOGICAL   CHEMISTRY 

of  wliich  the  temperature  of  the  fluid  under  examination  is  lowered. 
A  large  glass  tube  (B)  designed  as  an  air-jacket,  and  formed  after  the 
manner  of  a  test-tube  is  introduced  through  the  central  aperture  in  the 
metal  cover  and  into  this  air-jacket  is  lowered  a  smaller  tube  (A)  con- 
taining the  fluid  to  be  tested.  A  very  delicate  thermometer  (D),  grad- 
uated in  hundredths  of  a  degree  is  introduced  into  the  inner  tube  and 
is  held  in  place  by  means  of  a  cork  so  that  the  mercury  bulb  is  im- 
mersed in  the  fluid  under  examination  but  does  not  come  into  contact 
with  any  glass  surface.  A  small  platinum  wire  stirrer  serves  to  keep 
the  fluid  under  examination  well  mixed  while  a  larger  stirrer  is  used  to 
manipulate  the  freezing  mixture.  (Rock  salt  and  ice  in  the  proportion 
1 : 3  form  a  very  satisfactory  freezing  mixture.) 

In  making  a  determination  of  the  freezing-point  of  a  fluid  by  means 
of  the  Beckmann-Heidenhain , apparatus  proceed  as  follows:  Place 
the  freezing  mixture  in  the  battery  jar  and  add  water  (if  necessary)  to 
secure  a  temperature  not  lower  than  3°C.  Introduce  the  fluid  to  be 
tested  into  tube  A,  place  the  thermometer  and  platinum  wire  stirrer  in 
position,  and  insert  the  tube  into  the  air-jacket  which  has  previously 
been  inserted  through  the  metal  cover  of  the  battery  jar.  Manipulate 
the  two  stirrers  in  order  to  insure  an  equalization  of  temperature 
and  observe  the  course  of  the  mercury  column  of  the  thermometer  very 
carefully.  The  niercury  will  gradually  fall  and  this  gradual  lowering 
of  the  temperature  will  be  followed  by  a  sudden  rise.  The  point  at 
which  the  mercury  rests  after  this  sudden  rise  is  the  freezing-point. 
This  rise  is  due  to  the  fact  that  previous  to  freezing,  a  fluid  is  always 
more  or  less  over-cooled  and  the  thermometer  temporarily  registers  a 
temperature  somewhat  helow  the  freezing-point.  As  the  fluid  freezes, 
however,  there  is  a  very  sudden  change  in  the  temperature  of  the  liquid 
and  this  change  is  imparted  to  the  thermometer  and  causes  the  rise  as 
indicated.  It  occasionally  occurs  that  the  fluid  under  examination  is 
very  much  over-cooled  and  does  7iot  freeze.  Under  such  circumstances 
a  small  piece  of  ice  is  introduced  into  it  by  means  of  the  side  tube  noted 
in  the  figure.  This  so-called  "inoculation"  causes  the  fluid  to  freeze 
instantaneously.  (For  details  of  the  method  of  determining  the 
freezing-point  consult  standard  works  on  physical  or  organic  chemistry.) 

Electrical  Conductivity. — The  electrical  conductivity  of  the  urine 
is  dependent  upon  the  number  of  inorganic  molecules  or  ions  present, 
and  in  this  differs  from  the  freezing-point  which  is  dependent  upon  the 
total  number  of  molecules  both  inorganic  and  organic  which  are  in 
solution.  The  conductivity  of  the  urine  has  been  investigated  but 
slightly,  but  from  the  data  secured  it  seems  that  the  value  generally 
falls  below  k  =  o.o2,.-   The  conductivity  of  blood  serum  has  been  de- 


URINE  367 

termined  as  k  =  0.012.  Up  to  the  present  time  the  determination  of 
the  electrical  conductivity  of  any  of  the  fluids  of  the  body  has  been 
put  to  very  slight  clinical  use.  Experience  may  show  the  conductivity 
value  to  be  a  more  important  aid  to  diagnosis  than  it  is  now  considered, 
particularly  if  it  is  taken  in  connection  with  the  determination  of  the 
freezing-point.  By  a  combination  of  these  two  methods  the  portion 
of  the  osmotic  pressure  due  respectively  to  electrolytes  and  non- 
electrolytes  may  be  determined.  For  a  discussion  of  electrical  con- 
ductivity, the  method  by  which  it  is  determined,  and  the  principles 
involved  consult  standard  works  on  physical  or  electro-chemistry. 

Collection  and  Preservation  of  the  Urine  Sample. — If  any  depend- 
able data  are  desired  regarding  the  quantitative  composition  of  the  urine 
the  examination  of  the  mixed  excretion  for  twenty-four  hours  is  ab- 
solutely necessary.  In  collecting  the  urine  the  bladder  may  be  emptied 
at  a  given  hour,  say  8  A.  M.,  the  urine  discarded  and  all  the  urine  from 
that  hour  up  to  and  including  that  passed  the  next  day  at  8  A.  M., 
saved,  thoroughly  mixed,  and  a  sample  taken  for  analysis.  Until 
recently  it  was  believed  that  powdered  thymol  (isopropylmetacresol) 

CH3 


OH 

CHs — CH — CH3, 

was  a  very  satisfactory  preservative  since  the  excess  might  be  removed 
by  filtration,  if  desired,  and  it  was  believed  that  the  small  amount  which 
went  into  solution  would  have  no  appreciable  influence  upon  the  deter- 
mination of  any  of  the  urinary  constituents.  It  appears  however  that 
thymol  is  not  such  a  satisfactory  urinary  preservative  as  was  believed. 
Evidence  has  been  presented  showing  it  to  be  unsatisfactory  for  the 
preservation  of  urines  which  contain  sugar,  acetone  or  diacetic  acid 
and  in  which  it  desired  to  estimate  the  quantitative  content  of  these 
constituents.  Claim  has  also  been  made  that  thymol  is  not  a  satis- 
factory preservative  for  urines  that  are  to  be  examined  quantitatively  for 
phosphates  or  magnesium.  Thymol  being  a  phenol  will  cause  an  in- 
accuracy when  phenols  are  being  determined  quantitatively.  Urines 
preserved  by  thymol  will  also  give  a  confusing  white  ring  when  sub- 
jected to  the  nitric  acid  test  for  albumin  (see  Chapter  XXIII). 

Toluene  is  a  very  satisfactory  preservative  for  urine.     In  using  this 
preservative  simply  overlay  the  urine  with  the  toluene.     Rosenbloom^ 

*  Rosenbloom:  New  York  Medical  Journal,  99,  735,  1914. 


368  PHYSIOLOGICAL    CHEMISTRY 

claims  that  camphor  is  a  very  satisfactory  urine  preservative  which 
does  not  interfere  with  the  tests  for  important  urinary  constituents. 

In  certain  pathological  conditions  it  is  desirable  to  collect  the  urine 
passed  during  the  day  separately  from  that  passed  during  the  night. 
When  this  is  done  the  urine  voided  between  8  A.  M.  and  8  P.  M.  may 
be  taken  as  the  day  sample  and  that  voided  between  8  P.  M.  and  8  A.  M. 
as  the  night  sample. 

The  qualitative  testing  of  urine  samples  collected  at  random,  except 
in  a  few  specific  instances,  is  of  no  particular  value  so  far  as  giving  us 
any  accurate  knowledge  as  to  the  exact  urinary  characteristics  of  the 
individual  is  concerned.  In  the  great  majority  of  cases  the  qualitative 
as  well  as  the  quantitative  tests  should  be  made  upon  the  mixed 
excretion  for  a  twenty-four-hour  period  as  well  as  upon  a  night  sample 
as  ^above  described. 


CHAPTER  XXII 


URINE:  PHYSIOLOGICAL  CONSTITUENTS' 
I.  Organic  Physiological  Constituents 


Urea. 
Uric  acid. 
Creatinine. 
Creatine. - 

Ethereal  sulphuric  acids. 


f  Indoxyl-sulphuric  acid. 

J  Phenol-  and  /j-cresol-sulphuric- acids. 

j  Pyrocatechol-sulphuric  acid. 

I  Skatoxyl-sulphuric  acid. 


Hippuric  acid. 
Oxalic  acid. 


Neutral  sulphur  compounds. 


Allantoin. 


Aromatic  oxyacids. 


f  Cystine. 

Chondroitin-sulphuric  acid. 
:  Thiocyanates. 

Taurine  derivatives. 

Ox}'proteic  acid. 

Alloxj^Droteic  acid. 

Uroferric  acid. 

'  Para-oxyphenyl-acetic  acid. 
Para-oxyphenyl-propionic  acid. 
Homogentisic  acid. 
Uroleucic  acid. 
I  Oxymandelic  acid. 
I  Kynurenic  acid. 
Amino-acids. 
Peptides. 
Benzoic  acid. 
Nucleoprotein. 
Oxaluric  acid. 
Glucose. 

*  It  is  impossible  to  make  any  absolute  classification  of  the  physiological  and  pathological 
constituents  of  the  urine.  \  substance  may  be  present  in  the  urine  in  small  amount  physio- 
logically and  be  sufBciently  increased  under  certain  conditions  as  to  be  termed  a  patholog- 
ical constituent.  Therefore  it  depends,  in  some  instances  upon  the  quantity  of  a  constituent 
present  whether  it  may  be  correctly  termed  a  physiological  or  a  pathological  constituent. 

'  Normal  constituent  of  urine  of  adults  but  found  in  larger  amount  in  urine  of  infants 
and  children  (see  p.  509). 

24  369 


370 


PHYSIOLOGICAL    CHEMISTRY 


Pepsin. 

Enzymes Gastric  rennin. 

Amylase. 
[  Acetic  acid. 

Volatile  fatty  acids Butyric  acid.  • 

[  Formic  acid. 
Paralactic  acid. 
Phenaceturic  acid. 
Urocanic  acid. 

Phosphorized  compounds 


Pigments 

Ptomaines  and  leucomaines. 


Glycerophosphoric  acid. 
Phosphocarnic  acid. 
Urochrome. 
Urobilin. 
Uroerythrin. 


Adenine. 
Guanine. 
Xanthine. 
Epiguanine. 

Purine  Bases '  Episarkine. 

Hypoxanthine. 
Paraxanthine. 
Heteroxanthine. 
i-Methylxanthine. 

2.  Inorganic  Physiological  Constituents 

Ammonia. 

Sulphates. 

Chlorides. 

Phosphates. 

Sodium  and  potassium. 

Calcium  and  magnesium. 

Carbonates. 

Iron. 

Fluorides. 

Nitrates. 

Silicates. 

Hydrogen  peroxide. 

Normal  urine  varies  widely  in  composition,  being  influenced  by  diet 
and  other  factors.  The  following  table  represents  the  composition  of  a 
normal  urine. ^ 

^  Vierordt:  Daten  und  Tabellen.     Jena,  1906,  p.  330. 


URINE 


371 


COMPOSITION  OF  A  NORMAL  URINE 
Volume  (24  hours)  1500  c.c. 


Constituent 


""St"      ■    Approximate 
l^a^ms*      I      percentage 


Water '  1440.00 


Solids 


60.0 


Urea 


35-0 


Uric  acid 


0.75 


Hippuric  acid 


0.7 


Oxalic  acid 


0.015 


Aromatic  oxyacids 


0.06 


Creatinine 


Magnesium  (MgO) 


0.30 


96.0 


4.0 


2-Z2, 


0.004 


Thiocyanic  acid  (as  KSCN) 

0-15 

o.or 

Indican 

o.oi 

O.OOI 

Ammonia 

0.65 

0.04           1 

Sodium  chloride 

16.5 

I.I             i 

Phosphoric  acid 

2-5 

0-15 

Total  sulphuric  acid^ 

2-5 

1 

Silicic  acid 

0.45 

0.03 

Potassium  (K2O) 

2-5 

0.15           i 

1 

Sodium  (NajO) 

5-0 

0-3 

Calcium  (CaO) 

0.25 

0-015 

Iron • o .  005  o .  0004 

1  For  data  as  to  "partition"  of  sulphur  and  nitrogen,  see  Chapter  XXVII  on  Metabolism. 


372  PHYSIOLOGICAL    CHEMISTRY 

NHo 
UREA,  C  =  0. 

NHo 

Urea  is  the  principal  end-product  of  the  metaboHsm  of  protein 
substances.  It  was  formerly  believed  that  about  90  per  cent  of  the 
total  nitrogen  of  the  urine  was  present  as  urea.  Folin,  however,  has 
shown  that  the  distribution  of  the  nitrogen  of  the  urine  among 
urea  and  the  other  nitrogen-containing  bodies  present  depends  entirely 
upon  the  absolute  amount  of  the  total  nitrogen  excreted.  He  found 
that  a  decrease  in  the  total  nitrogen  excretion  was  always  accom- 
panied by  a  decrease  in  the  percentage  of  the  total  nitrogen  excreted 
as  urea,  and  that  after  so  regulating  the  diet  of  a  normal  person  as  to^ 


Fig.  118. — Urea. 

cause  the  excretion  of  total  nitrogen  to  be  reduced  to  3-4  grams  in  24 
hours,  only  about  60  per  cent  of  this  nitrogen  appeared  in  the  urine  as  urea. 
His  experiments  also  seem  to  show  urea  to  be  the  only  one  of  the  nitro- 
genous excretions  which  is  relatively  as  well  as  absolutely  decreased  as  a 
result  of  decreasing  the  amount  of  protein  metabolized.  This  same 
investigator  reports  a  hospital  case  in  which  only  14.7  per  cent  of  the 
total  nitrogen  was  present  as  urea  and  about  40  per  cent  was  present  as 
ammonia.  Morner  had  p'reviously  reported  a  case  in  which  but  4.4 
per  cent  of  the  total  nitrogen  of  the  urine  was  present  as  urea,  and  26.7 
per  cent  was  present  as  ammonia. 

Urea  occurs  most  abundantly  in  the  urine  of  man  and  carnivora 
and  in  somewhat  smaller  amount  in  the  urine  of  herbivora;  the  urine 
of  fishes,  amphibians,  and  certain  birds  also  contains  a  small  amount  of 


URINE 


373 


the  substance.  Urea  is  also  found  in  nearly  all  the  fluids  and  in  many 
of  the  tissues  and  organs  of  mammals.  The  amount  excreted,  under 
normal  conditions,  by  an  adult  man  in  24  hours  is  about  30-35  grams. 
The  excretion  is  greatest  in  amount  after  a  diet  of  meat,  and  least  in 
amount  after  a  diet  consisting  of  non-nitrogenous  foods;  this  is  due  to  the 
fact  that  the  urea  output  is  regulated  by  the  protein  ingestion.  It  is 
true  also  that  a  non-nitrogenous  diet  has  a  tendency  to  decrease  the 
metabolism  of  the  tissue  proteins  and  thus  cause  the  output  of  urea  under 
these  conditions  to  fall  below  the  output  of  urea  observed  during  starva- 
tion. The  output  of  urea  is  also  increased  after  copious  water-  or  beer- 
drinking.  The  increase  is  probably  due  primarily  to  the  washing  out  of 
the  tissues  of  the  urea  previously  formed,  but  which  had  not  been  re- 
moved in  the  normal  processes,  and  secondarily  to  a  stimulation  of 
protein  catabolism. 

Urea  may  be  formed  in  the  organism  from  amino-acids  such  as  leu- 
cine, glycocoll,  and  aspartic  acid :  it  may  also  be  formed  from  ammonium 
carbonate  (NH4)2C03  or  ammonium  carbamate,  H4X.O.CO.XH2. 

There  are  differences  of  opinion  regarding  the  transformation  of  the 
substances  just  named  into  urea,  but  there  is  rather  conclusive  e\ddence 
that  at  least  a  part  of  the  urea  is  formed  in  the  liver;  it  may  be  formed  in 
other  organs  or  tissues  as  well. 

Urea  crystallizes  in  long,  colorless,  four-  or  sLx-sided,  anhydrous, 
rhombic  prisms  (Fig.  118),  which  melt  at  i32°C.  and  are  soluble  in 
water  or  alcohol  and  insoluble  in  ether  or  chloroform.  If  a  crystal  of 
urea  is  heated  in  a  test-tube,  it  melts  and  decomposes  with  the  liberation 
of  ammonia.     The  residue  contains  cyanuric  acid, 

C.OH 

N      N 

II       I 
HO.C      C.OH 

N 

NH2 

C  =  0 

and  biuret,  \ 

NH 

/ 
C  =  0 

I 
NH2 


374 


PHYSIOLOGICAL   CHEMISTRY 


The  biuret  may  be  dissolved  in  water  and  a  reddish-violet  color  obtained 
by  treating  the  aqueous  solution  with  copper  sulphate  and  potassium 
hydroxide  (see  Biuret  Test,  page  98).  Certain  hypochlorites  or  hypo- 
bromites  in  alkaline  solution  have  the  power  of  decomposing  urea  into 
nitrogen,  carbon  dioxide,  and  water.  Sodium  hypobromite  brings 
about  this  decomposition,  as  follows: 

CO(NH2)2+3NaOBr-»3NaBr+N2+C02+2H20. 

This  property  forms  the  basis  for  a  clinical  quantitative  determination 
of  urea  (see  page  496). 

The  soy  bean  has  been  shown  to  contain  an  enzyme  called  urease 
which  has  the  power  to  decompose  urea  with  the  liberation  of  ammonia.^ 
This  fact  is  made  use  of  in  the  quantitative  determination  of  urea 
(see  Chapter  XXVI). 


Fig.  119. — Urea  Nitrate. 


Urea  has  the  power  of  forming  crystalline  compounds  with  certain 
acids;  urea  nitrate  and  urea  oxalate  are  the  most  important  of  these 
compounds.  Urea  nitrate,  CO(NH2)2.HN03,  crytallizes  in  colorless, 
rhombic  or  six-sided  tiles  (Fig.  119,  above),  which  are  easily  soluble  in 
water.  Urea  oxalate,  [CO(NH2)2]2-H2C204,  crystallizes  in  the  form 
of  rhombic  or  six-sided  prisms  or  plates  (Fig.  121,  page  376):  the 
oxalate  differs  from  the  nitrate  in  being  somewhat  less  soluble  in 
water.  The  formation  of  the  nitrate  and  oxalate  and  the  decomposition 
of  urea  by  the  enzyme  urease  are  the  most  satisfactory  methods  for  the 
detection  of  urea. 

A  decrease  in  the  excretion  of  urea  is  observed  in  many  diseases  in 
which  the  diet  is  much  reduced  and  in  some  disorders  as  a  result  of 

^Takeuchi:  Jour.  College  of  Agr.,  Tokyo,  1909,  Part  I. 


URINE 


375 


alterations  in  metabolism,  e.g.,  myxedema,  and  in  others  as  a  result 
of  changes  in  excretion,  as  in  severe  and  advanced  kidney  disease.  A 
pathological  increase  is  found  in  a  large  proportion  of  diseases  which 
are  associated  with  a  toxic  state.  In  marked  acidosis  it  may  be  con- 
siderably decreased  relative  to  the  total  nitrogen  (see  Ammonia). 


Experiments  on  Urea 

1.  Isolation  from  the  Urine.  ^ — Place  800  c.c.  of  urine  in  a  precipitating  jar, 
add  250  c.c.  of  baryta  inixture,^  and  stir  thoroughly.  Filter  off  the  precipitate 
of  phosphates,  sulphates,  ixrates,  and  hippurates  and 
evaporate  the  filtrate  on  a  water-bath  to  a  thick  syrup. 
This  syrup  contains  chlorides,  creatinine,  organic  salts, 
pigments,  and  urea.  Extract  the  syrup  with  warm  95 
per  cent  alcohol  and  filter  again.  The  filtrate  con- 
tains the  ixrea  contaminated  with  pigment.  Decolor- 
ize the  filtrate  by  boiUng  with  animal  charcoal,  filter 
again,  and  stand  the  filtrate  away  in  a  cold  place  for 
crystallization.  Examine  the  crystals  under  the  micro- 
scope and  compare  them  with  those  shown  in  Fig.  118, 
page  372. 

2.  Solubility. — Test  the  solubiUty  of  urea,  prepared 
by  yourself  or  furnished  by  the  instructor,  in  water  and 
in  alcohol  and  ether. 

3.  Melting-point. — Determine  the  melting-point  of 
some  pure  urea  furnished  by  the  instructor.  Proceed 
as  follows:  Into  an  ordinary  melting-point  tube,  sealed 
at  one  end,  introduce  powdered  urea.  Fasten  the  tube 
to  the  bulb  of  a  thermometer  as  shown  in  Fig.  120,  and 
suspend  the  bulb  and  its  attached  tube  in  a  small  beaker 
containing  sulphuric  acid.  Gently  raise  the  tempera- 
ture of  the  acid  by  means  of  a  low  flame,  stirring  the 
fluid  continually,  and  note  the  temperature  at  which 
the  urea  begins  to  melt. 

4.  Crystalline  Form. — ^Dissolve  a  crystal  of  pure 
urea  in  a  few  drops  of  95  per  cent  alcohol  and  place 
1-2  drops  of  the  alcohoUc  solution  on  a  microscopic 
slide.     Allow  the  alcohol  to  evaporate  spontaneously, 

examine  the  crystals  under  the  microscope,  and  compare  them  with  those  re- 
produced in  Fig,  n8,  page  372.  RecrystaUize  a  Uttle  urea  from  water  in  the 
same  way  and  compare  the  crystals  with  those  obtained  from  the  alcohoUc 
solution. 

5.  Formation  of  Biuret. — Place  a  small  amount  of  urea  in  a  dry  test-tube 
and  heat  carefully  in  a  low  flame.  The  lu-ea  melts  at  I32°C.  and  Uberates 
ammonia.     Continue  heating  until  the  fused  mass  begins  to  solidify.     Cool  the 

^  The  method  based  upon  tlie  precipitation  by  nitric  acid  is  also  satisfactory'  (see 
Hoppe-Seyler's  Handbuch dcr  Physiol,  und Pathol.  Chem.  Anal.,  Eighth  edition,  1909,  p.  145). 

'  Baryta  mbcture  consists  of  a  mixture  of  i  volume  of  a  saturated  solution  of  Ba(NOj)j 
and  2  volumes  of  a  saturated  solution  of  Ba(OH)j. 


Fig.  120. — ]Melting- 
poiNT  Tubes  Fastened 
TO  Bulb  of  Thermom- 
eter. 


376 


PHYSIOLOGICAL    CHEMISTRY 


tube,  dissolve  the  residue  in  dilute  potassium  hydroxide  solution,  and  add  very 
dilute  copper  sulphate  solution  (see  page  98).  The  purpUsh -violet  color  is  due 
to  the  presence  of  biuret  which  has  been  formed  from  the  urea  through  the 
application  of  heat  as  indicated.     This  is  the  reaction : 


NH.2 

I 

Urea.   C  =  O 

\ 

N 

/< 

Urea,   C  =0 

NH, 


H 


H 


NH2 

C=0 

\ 
NH  +  NH3 

/ 
C=0 

NH2 

Biuret. 


6.  Urea  Nitrate. — Prepare  a  concentrated  solution  of  urea  by  dissolving 
a  little  of  the  substance  in  a  few  drops  of  water.  Place  a  drop  of  this  solution  on  a 
microscopic  slide,  add  a  drop  of  concentrated  nitric  acid,  and  examine  under 
the  microscope.     Compare  the  crystals  with  those  reproduced  in  Fig.  119,  page  374. 


Fig.  121.— Urea  Oxalate. 

7.  Urea  Oxalate. — To  a  drop  of  a  concentrated  solution  of  urea,  prepared  as 
described  in  the  last  experiment  (6),  add  a  drop  of  a  saturated  solution  of  oxalic 
acid.  Examine  under  the  microscope  and  compare  the  crystals  with  those  shown 
in  Fig.  121,  above. 

8.  Decomposition  by  Sodium  Hypobromite. — Into  a  mixture  of  3  c.c.  of  con- 
centrated sodiimi  hydroxide  solution  and  2  c.c.  of  bromine  water  in  a  test-tube 
introduce  a  crystal  of  urea  or  a  small  amount  of  concentrated  solution  of  urea. 
Through  the  influence  of  the  sodium  hypobromite,  NaOBr,  the  urea  is  decom- 
posed and  carbon  dioxide  and  nitrogen  are  hberated.  The  carbon  dioxide  is 
absorbed  by  the  excess  of  sodium  hydroxide,  while  the  nitrogen  is  evolved  and 
causes  the  marked  effervescence  observed.     This  property  forms  the  basis  for 


URINE  .577 

one  of  the  methods  in  common  use  for  the  quantitative  determination  of  urea. 
Write  the  equation  showing  the  decomposition  of  urea  by  sodium  hypobromite. 

9.  Furfural  Test. — To  a  few  crystals  of  urea  in  a  small  porcelain  dish  add  1-2 
drops  of  a  concentrated  aqueous  solution  of  furfural  and  1-2  drops  of  concentrated 
hydrochloric  acid.  Note  the  appearance  of  a  yellow  color  which  gradually  changes 
into  a  purple.     Allantoin  also  responds  to  this  test  (see  page  392). 

It  is  claimed  that  all  ammonium  compounds  and  all  compounds 
containing  the  amino  (—  NH2)  group  yield  nitrogen  when  treated  with 
hypobromite  as  in  this  test. 

HN— CO 

URIC  ACID,   OC      C-NH 

>C0. 
HN— C— HN 

Uric  acid  is  one  of  the  most  important  of  the  constituents  of  the 
urine.  It  is  generally  stated  that  normally  about  0.7  gram  is  excreted 
in  24  hours,  but  that  this  amount  is  subject  to  wide  variations,  particu- 
larly under  certain  dietary  and  pathological  conditions.  It  has  been 
shown,  however,  that  the  average  daily  excretion  of  uric  acid  for  ten 
men  ranging  in  age  from  19  to  29  years  and  fed  a  normal  mixed  diet 
was  0.597  gram,  a  value  somewhat  lower  than  the  generally  accepted 
average  of  0.7  gram  for  such  a  period.  On  a  purine-free  diet  the  uric 
acid  output  may  be  o.  1-0.5  gram  per  day,  whereas  a  high  purine  diet  may 
yield  a  daily  output  of  2  grams.  Uric  acid  is  a  diureide  and  consequently 
upon  oxidation  may  yield  two  molecules  of  urea.  It  acts  as  a  weak 
dibasic  acid  and  forms  two  classes  of  salts,  neutral  and  acid.  The  neutral 
potassium  and  lithium  urates  are  the  most  easily  soluble  of  the  alkali 
salts;  the  ammonium  urate  is  difficultly  soluble.  The  acid-alkali  urates 
are  more  insoluble  and  form  the  major  portion  of  the  sediment  which 
separates  upon  cooling  the  concentrated  urine;  the  alkaline  earth  urates 
are  very  insoluble.  Ordinarily  uric  acid  occurs  in  the  urine  in  the  form 
of  urates  and  upon  acidifying  the  liquid  the  uric  acid  is  liberated  and 
deposits  in  crystalline  form.  This  property  forms  the  basis  of  one  of 
the  older  methods  for  the  quantitative  determination  of  uric  acid 
(Heintz  jMcthod,  Chapter  XXVI). 

Uric  acid  is  very  closely  related  to  the  purine  bases  as  may  be  seen 
from  a  comparison  of  its  structural  formula  with  those  of  the  purine 
bases  given  on  page  127.  According  to  the  purine  nomenclature  it  is 
designated  2-6-8-trioxypurine.  Uric  acid  forms  the  principal  end- 
product  of  the  nitrogenous  metabolism  of  birds  and  scaly  amphibians; 
in  the  human  organism  it  occupies  the  fourth  position  inasmuch  as  here 


37^  PHYSIOLOGICAL   CHEMISTRY 

urea,  ammonia,  and  creatinine  are  the  chief  end-products  of  nitroge- 
nous metabolism.  It  is  generally  said  that  the  relation  existing  between 
uric  acid  and  urea  in  human  urine  under  normal  conditions  varies  on 
the  average  from  i :  40  to  i :  100  and  is  subject  to  wider  variations  under 
pathological  conditions;  and  further  that  because  of  the  high  content  of 
uric  acid  in  the  urine  of  newborn  infants  the  ratio  may  be  reduced  to 
1 :  10  or  even  lower.  We  now  know  that  this  ratio  of  uric  acid  to  urea 
is  of  little  significance  under  any  conditions. 

In  man,  uric  acid  probably  results  principally  from  the  destruction 
of  nuclein  material.  It  may  arise  from  nuclein  or  other  purine  material 
ingested  as  food  or  from  the  disintegrating  cellular  matter  of  the  organ- 
ism. The  uric  acid  resulting  from  the  first  process  is  said  to  be  of  ex- 
ogenous origin,  whereas  the  product  of  the  second  form  of  activity  is 
said  to  be  of  endogenous  origin.  As  a  result  of  experimentation,  Siven, 
and  Burian  and  Schur,  and  Rockwood  claim  that  the  amount  of  endoge- 
nous uric  acid  formed  in  any  given  period  is  fairly  constant  for  each 
individual  under  normal  conditions,  and  that  it  is  entirely  independent 
of  the  total  amount  of  nitrogen  eliminated.  Folin  has  taken  exception 
to  the  statements  of  these  investigators  and  claims  that,  following  a 
pronounced  decrease  in  the  amount  of  protein  metabolized,  the  absolute 
quantity  of  uric  acid  is  decreased  but  that  this  decrease  is  relatively 
smaller  than  the  decrease  in  the  total  nitrogen  excretion  and  that  the 
per  cent  of  the  uric  acid  nitrogen,  in  terms  of  the  total  nitrogen,  is  there- 
fore decidedly  increased.  According  to  Mares, ^  food-stuffs  act  to  in- 
crease the  endogenous  uric  acid  output  by  stimulating  the  digestive 
glands  to  activity.  That  a  portion  of  the  endogenous  uric  acid  may 
arise  in  this  way  has  recently  been  shown  by  Mendel  and  Stehle.^ 

In  birds  the  formation  of  uric  acid  is  analogous  to  the  formation 
of  urea  in  man.  In  these  organisms  it  is  derived  principally  from  the 
protein  material  of  the  tissues  and  the  food  and  is  formed  through  a 
process  of  synthesis  which  occurs  for  the  most  part  in  the  liver;  a 
comparatively  small  fraction  of  the  total  uric  acid  excretion  of  birds 
may  result  from  nuclein  material. 

When  pure,  uric  acid  may  be  obtained  as  a  white,  odorless,  and 
tasteless  powder,  which  is  composed  principally  of  small,  transparent, 
crystalline,  rhombic  plates.  Uric  acid  as  it  separates  from  the  urine 
is  invariably  pigmented,  and  crystallizes  in  a  large  variety  of  character- 
istic forms,  e.g.,  dumb-bells,  wedges,  rhombic  prisms,  irregular  rect- 
angular or  hexagonal  plates,  whetstones,  prismatic  rosettes,  etc.  Uric 
acid  is  insoluble  in  alcohol  and  ether,  soluble  with  difficulty  in  boiling 

^  Mares:  Arch.  f.  d.  ges.  Physiol.,  134,  59,  1910. 

*  Mendel  and  Stehle:  Jour.  Biol.  Chem.,  22,  215,  1915. 


URINE  379 

water  (i  :i8oo)  and  practically  insoluble  in  cold  water  (i  : 39,480.  at 
i8°C.).  It  is  soluble  in  alkalis,  alkali  carbonates,  boiling  glycerol., 
concentrated  sulphuric  acid,  and  in  certain  organic  bases  such  as  ethyl- 
amine  and  piperidine.  It  is  claimed  that  the  uric  acid  is  held  in  solu- 
tion in  the  urine  by  the  urea  and  disodium  hydrogen  phosphate  present. 
Uric  acid  possesses  the  power  of  reducing  cupric  hydroxide  in  alkaline 
solution  and  may  thus  lead  to  an  erroneous  conclusion  in  testing  for 
sugar  in  the  urine  by  means  of  Fehling's  or  Trommer's  test.  A  white 
precipitate  of  cuprous  urate  is  formed  if  only  a  small  amount  of  cupric 
hydroxide  is  present,  but  if  enough  of  the  copper  salt  is  present  the 
characteristic  red  or  brownish-red  precipitate  of  cuprous  oxide  is  ob- 
tained. Uric  acid  does  not  possess  the  power  of  reducing  bismuth  in 
alkaline  solution  and  therefore  does  not  interfere  in  testing  for  sugar  in 
the  urine  by  means  of  Boettger's  or  Xylander's  tests. 

In  addition  to  being  an  important  urinary  constituent  uric  acid  is 
normally  present  in  the  brain,  heart,  Uver,  lungs,  pancreas,  and  spleen; 
it  also  occurs  in  the  blood  of  birds  and  has  been  detected  in  traces  in 
human  blood  under  normal  conditions. 

Pathologically,  the  excretion  of  uric  acid  is  subject  to  wide  varia- 
tions, but  the  experimental  findings  are  rather  contradictory.  It  may  be 
stated  with  certainty,  however,  that  in  leukemia,  because  of  the  destruc- 
tion of  nuclein  material,  the  uric  acid  output  is  increased  absolutely  as 
well  as  relatively  to  the  urea  output;  under  these  conditions  the  ratio 
between  the  uric  acid  and  uirea  may  be  as  low  as  i :  9,  whereas  the  normal 
ratio,  as  we  have  seen,  is  i :  50  or  higher.  An  actual  output  of  12  grams 
of  uric  acid  per  day  has  been  reported  in  leukemia.  In  the  study  of  the 
influence  of  X-ray  on  metabolism  Edsall  and  others  have  reached  some 
interesting  conclusions.  Edsall  found  that  the  excretion  of  uric  acid  is 
usually  increased  and  that  in  some  conditions,  particularly  in  leukemia, 
it  may  be  greatly  increased.  The  excretion  of  total  nitrogen,  phos- 
phates, and  other  sustances  may  also  be  considerably  increased. 

In  gout  the  kidney  is  said  to  lose  the  power  of  properly  eliminating 
uric  acid  and  it  collects  in  the  blood  in  abnormally  high  concentration. 

Normal  =  1-3  mg.  uric  acid  per  100  grams  of  blood. 
Gout  =  3-6  mg.  uric  acid  per  100  grams  of  blood. 

In  gout  the  uric  acid  content  of  the  urine  is  generally  low  preceding 
an  attack  and  increases  during  the  attack.  Atophan  has  been  found 
to  increase  the  uric  acid  output  in  gout,  apparently  due  to  increased 
kidney  activity. 

The  uric  acid  content  of  the  urine  is  of  importance  in  relation  to  the 
formation  of  uric  acid  calculi.     The  administration  of  alkali  carbonates 


380  PHYSIOLOGICAL    CHEMISTRY 

and  citrates,  or  the  feeding  of  base-forming  foods,  by  decreasing  the 
acidity  of  the  urine  increases  its  solvent  power  for  uric  acid  and  de- 
creases the  liability  of  formation  of  this  type  of  calculus.^ 

Experiments  on  Uric  Acid 

1.  Isolation  from  the  Urine. — Place  about  200  c.c.  of  filtered  urine  in  a 
beaker,  render  it  acid  with  2-10  c.c.  of  concentrated  hydrochloric  acid,  stir 
thoroughly,  and  stand  the  vessel  in  a  cold  place  for  24  hours.  Examine  the  pig- 
mented crystals  of  uric  acid  under  the  microscope  and  compare  them  with  those 
shown  in  Fig.  136,  page  461 ,  and  PI.  V,  opposite. 

2.  Solubility. — Try  the  solubiUty  of  pure  uric  acid,  furnished  by  the  in- 
structor, in  water,  dilute  acid  and  alkaU  and  in  alcohol,  ether  and  concentrated 
sulphuric  acid. 

3.  Crystalline  Form  of  Pure  Uric  Acid. — Place  about  100  c.c.  of  water  in  a 
small  beaker,  render  it  distinctly  alkaUne  with  potassivun  hydroxide  solution  and 


Fig.  122. — Pure  Uric  Acid. 

add  a  small  amount  of  pure  vuic  acid,  stirring  continuously.  Cool  the  solution, 
render  it  distinctly  acid  with  hydrochloric  acid  and  allow  it  to  stand  in  a  cool 
place  for  crystallization.  Examine  the  crystals  under  the  microscope  and  com- 
pare them  with  those  reproduced  in  Fig.  122. 

4.  Murexide  Test. — To  a  small  amount  of  pure  uric  acid  in  a  small  evaporating 
dish  add  2-3  drops  of  concentrated  nitric  acid.  Evaporate  to  dryness  carefully 
on  a  water-bath  or  over  a  very  low  flame.  A  red  or  yellow  residue  remains  which 
turns  purpUsh  red  after  cooUng  the  dish  and  adding  a  drop  of  very  dilute  am- 
monium hydroxide.  The  color  is  due  to  the  formation  of  murexide.  If  potas- 
sium hydroxide  is  used  instead  of  ammonium  hydroxide  a  purpUsh  violet  color 
due  to  the  production  of  the  potassium  salt  is  obtained.  The  color  disappears 
upon  warming ;  with  certain  related  bodies  (purine  bases)  the  color  persists  under 
these  conditions. 

^  Blather  wick:  Arch.  Int.  Med.,  14,  409,  1914. 


PLATE  V. 


Uric  Acid  Crystals.     Normal  Color.     (From  Pnrdy,  after  Fryer.) 


URINE  381 

In  this  reaction  the  uric  acid  is  oxidized  to  dialuric  acid  and  alloxan. 
These  two  substances  condense  to  form  alloxantin.  This  alloxantin 
reacts  with  ammonium  hydroxide  to  form  purpuric  acid.  The  purple 
color  is  due  to  the  formation  of  ammonium  piirpurate  or  miirexide. 

5.  Phosphotungstic  Acid  Reaction  (Folin). — To  20  c.c.  of  saturated  sodium 
carbonate  solution  in  a  small  beaker  add  a  small  amount  of  uric  acid.  Stir 
the  solution  until  the  uric  acid  has  dissolved,  then  add  i  c.c.  of  FoUn's  uric 
acid  reagent  (see  Chapter  XXVI 1.     A  blue  color  results. 

6.  Silver  Reduction  Test  fSchiff  1. — Dissolve  a  small  amount  of  pure  uric  acid 
in  sodium  carbonate  solution  and  transfer  a  drop  of  the  resulting  mixture  to  a 
strip  of  filter  paper  saturated  with  silver  nitrate  solution.  A  yellowish-brown 
or  black  coloration  due  to  the  formation  of  reduced  silver  is  produced. 

It  is  claimed  that  chlorides  interfere  with  this  test. 

7.  Ganassini's  Test.^ — Dissolve  a  small  amount  of  uric  acid  in  sodium  carbon- 
ate. Precipitate  the  dissolved  uric  acid  by  means  of  zinc  chloride,  filter  off  the 
precipitate,  and  permit  it  to  stand  in  contact  with  the  air.  A  sky-blue  color  will 
develop,  a  color  change  which  may  be  hastened  by  sunlight.  A  similar  reaction 
may  be  obtained  by  treating  the  original  precipitate  with  KiSjOg. 

8.  Influence  upon  Fehling's  Solution. — Dilute  i  c.c.  of  Fehling's  solution 
with  4  c.c.  of  water  and  heat  to  boiling.  Now  add  slowly,  a  few  drops  at  a 
time,  12  c.c.  of  a  concentrated  solution  of  uric  acid  in  potassium  hydroxide, 
heating  after  each  addition.  From  this  experiment  what  do  you  conclude  re- 
garding the  possibiUty  of  arriving  at  an  erroneous  decision  when  testing  for 
sugar  in  the  urine  by  means  of  Fehling's  test? 

9.  Reduction  of  Nylander's  Reagent. — To  5  c.c.  of  a  solution  of  uric  acid 
in  potassium  hydroxide  add  about  one-half  a  cubic  centimeter  of  Nylander's 
reagent  and  heat  to  boiUng  for  a  few  moments.  Do  you  obtain  the  typical  black 
end-reaction  signifying  the  reduction  of  the  bismuth? 

NH CO 

'  I 

CREATININE,  C  =  XH      I 

N.(CH3).CH2. 

Creatinine  is  the  anhydride  of  creatine  and  is  a  constituent  of  normal 
human  urine.  The  theory  that  creatinine  is  derived  from  the  creatine 
of  ingested  muscular  tissue  as  well  as  from  the  creatine  of  the  muscular 
tissue  of  the  organism  has  been  proven  to  be  incorrect  by  Folin, 
Klercker.  and  Wolf  and  Shaffer.  Shaffer  believes  that  creatinine  is 
the  result  of  some  special  process  of  normal  metabolism  which  takes 
place  to  a  large  extent,  if  not  entirely,  in  the  muscles,  and  further  that 
the  amount  of  such  creatinine  elimination,  expressed  /;/  milligrams  per 
kilogram  body  weight,  is  an  index  of  this  special  process.-     He  further 

'  Ganassini:  Boll,  svc,  1908,  No.  i. 

-He  proposes  to  designate  as  the  "creatinine  coefficient"  the  excretion  of  crcatininc- 
nilrogcn  {mg.)  per  kilogram  of  body  weight. 


382 


PHYSIOLOGICAL    CHEMISTRY 


states  that  the  muscular  efficiency  of  the  individual  depends  upon  the 
intensity  of  this  process.  Under  normal  conditions  about  i-i)-^  gram 
of  creatinine  is  excreted  by  an  adult  man  in  24  hours, ^  the  exact  amount 
depending  in  great  part  upon  the  nature  of  the  food  and  decreasing 
markedly  in  starvation.  Very  little  that  is  important  is  known  regard- 
ing the  excretion  of  creatinine  under  pathological  conditions.  The 
creatinine  content  of  the  urine  is  said  to  be  increased  in  typhoid  fever, 
typhus,  tetanus,  and  pneumonia,  and  to  be  decreased  in  anaemia,  chloro- 
sis, paralysis,  muscular  atrophy,  advanced  degeneration  of  the  kidneys, 
and  in  leukemia  (myelogeneous,  lymphatic  and  pseudo).     An  increase 


Fig.  123. — Creatinine. 

of  creatinine  was  also  noted  in  diabetes,  an  increase  probably  due  to  the 
creatinine  content  of  the  meat  eaten.  The  greater  part  of  the  data, 
however,  relating  to  the  variation  of  the  creatinine  excretion  under 
pathological  conditions  are  not  of  much  value  since  in  nearly  every 
instance  the  diet  was  not  sufficiently  controlled  to  permit  the  collection 
of  reliable  data.  And  further,  until  the  advent  of  the  Folin  method 
(see  page  506)  there  was  no  accurate  method  for  the  quantitative 
determination  of  creatinine.  Shaffer  has  called  attention  to  the  fact 
that  a  low  excretion  of  creatinine  is  found  in  the  urine  of  a  remarkably 
large  number  of  pathological  subjects,  representing  a  variety  of  con- 
ditions, and  that  it  is  therefore  evident  that  the  excretion  of  an  ab- 
normally small  amount  of  this  substance  is  by  no  means  peculiar  to  any 
one  disease.  A  considerable  increase  in  the  creatinine  content  of  the 
blood  has  been  observed  in  uremia.^ 

^  According  to  Shaffer  the  amount  excreted  by  strictly  normal  individuals  is  between  7 
and  II  mg.  of  creatinine-nitrogen  per  kilogram  of  body  weight. 
^  Folin  and  Denis:  Jotir.  Biol.  Client.,  17,  487,  1914. 
Myers  and  Fine:  Jotir.  Biol.  Chan.,  20,  391,  1914. 


URINE  383 

Creatinin  ecrystallizes  in  colorless,  glistening  monoclinic  prisms  (Fig. 
123,  page  382)  which  are  soluble  in  about  12  parts  of  cold  water;  they 
are  more  soluble  in  warm  water  and  in  warm  alcohol.  It  forms  salts  only 
with  strong  mineral  acids.  One  of  the  most  important  and  interesting 
of  the  compounds  of  creatinine  is  creatinme-zinc  chloride,  (04117X30)2- 
ZnCl2,  which  is  formed  from  an  alcoholic  solution  of  creatinine  upon 
treatment  with  zinc  chloride  in  acid  solution.  Creatinine  has  the  power 
of  reducing  cupric  hydroxide  in  alkaline  solution  and  in  this  way  may 
interfere  with  the  determination  of  sugar  in  the  urine.  In  the  reduction 
by  creatinine  the  blue  liquid  is  first  changed  to  a  yellow,  and  the  forma- 
tion of  a  brownish-red  precipitate  of  cuprous  oxide  is  brought  about  only 
after  continuous  boiling  with  an  excess  of  the  copper  salt.  Creatinine 
does  not  reduce  alkaline  bismuth  solutions  and  therefore  does  not  inter- 
fere with  Xylander's  and  Boettger's  tests. 

It  has  recently  been  shown  by  Folin  that  the  absolute  quantity  of 
creatinine  eliminated  in  the  urine  on  a  meat-free  diet  is  a  constant 
quantity  different  for  different  individuals,  but  wholly  independent  of 
quantitative  changes  in  the  total  amount  of  nitrogen  eliminated. 
Shaffer  has  very  recently  confirmed  these  findings  and  has  shown  that 
the  output  of  creatinine  under  these  conditions  is  constant  from  hour 
to  hour  as  well  as  from  day  to  day. 

Experiments  on  Creatinine 

i 

I.  Preparation  of  Pure  Creatmine  from  Urine  CFolin-Benedict^. — To  10  liters^ 
of  undecomposed  urine  in  a  large  precipitating  jar  add  with  stirring  a  hot  solution 
of  180  grams  of  picric  acid  in  450  c.c.  of  boiling  alcohol.  Allow  to  stand  over  night 
and  syphon  off  the  supernatant  fluid.  Pour  the  residue  upon  a  large  Buchner 
funnel,  drain  with  suction,  wash  once  or  twice  with  cold  saturated  picric  acid  and 
suck  dry.  Treat  the  dry  or  nearly  dry  picrate  in  a  large  mortar  or  evaporating 
dish  with  enough  concentrated  HCl  to  form  a  moderately  thin  paste  (about  60  c.c. 
of  acid  for  each  100  grams  of  picrate)  and  stir  the  mixture  thoroughly  with  the 
pestle  for  3-5  minutes.  Filter  with  suction  on  a  hardened  paper,  and  wash  the 
residue  twice  with  enough  water  to  cover  it,  sucking  as  nearly  dry  as  possible 
each  time.  Transfer  the  filtrate  to  a  large  flask  and  neutraUze  with  an  excess  of 
solid  magnesiimi  oxide  (the  "heavy"  variety  is  best).  Add  this  oxide  in  small 
portions  with  cooling  of  the  flask  under  running  water  between  the  additions. 
Neutralization  of  the  acid  will  be  indicated  by  a  bright  yellow  color  of  the  mix- 
txire,  or  litmus  paper  may  be  used  to  test  it.  Filter  with  suction.  Wash  the 
residue  twice  with  water.  Immediately  add  a  few  cubic  centimeters  of  glacial 
acetic  acid  to  the  filtrate  to  make  it  strongly  acid.  Pay  no  attention  to  any 
precipitate  that  may  form,  but  dilute  the  solution  with  about  4  volumes  of  95  per 

'  Benedict:  Jour.  Biol.  Clum.,  iS,  1S2,  1914. 

Folin:  Ibid.,  17   463,  1914. 
2  If  it  is  simply  desired  to  demonstrate  the  presence  of  creatinine,  i  liter  may  be  em- 
ployed and  the  various  reagents  reduced  accordingly. 


384  PHYSIOLOGICAL   CHEMISTRY 

cent  alcohol.  After  15  minutes  filter  off  the  sUght  precipitate  which  forms. 
Treat  the  final  filtrate  with  30-40  c.c.  of  30  per  cent  zinc  chloride.  Stir  and  let 
stand  over  night  in  a  cool  place.  Pour  off  the  supernatant  Uquid  and  collect  the 
creatinine  zinc  chloride  on  a  Buchner  funnel,  wash  once  with  water,  then  thor- 
oughly with  50  per  cent  alcohol,  finally  with  95  per  cent  alcohol  and  dry.  A 
nearly  white,  hght  crystalline  powder  should  be  obtained.  The  yield  should  be 
90-95  per  cent  of  the  original  creatinine  (usually  about  1.5-1.8  grams  of  creatinine 
zinc  chloride  per  Uter  of  urine). 

RecrystaUize  the  creatinine -zinc  chloride  by  treating  10  grams  with  100  c.c.' 
of  water  and  about  60  c.c.  of  normal  sulphuric  acid,  heating  the  mixture  until  a 
clear  solution  is  obtained.  Add  about  4  grams  of  purified  animal  charcoal,  con- 
tinue boiUng  for  about  a  minute,  filter  with  suction  through  a  small  Buchner 
funnel,  pouring  the  filtrate  back  on  the  filter  three  or  four  times  until  it  runs 
through  perfectly  colorless.  Wash  residue  with  hot  water  and  transfer  the  total 
filtrate  to  a  beaker  and  while  hot  treat  with  a  Uttle  strong  zinc  chloride  solution 
(3  c.c.)  and  with  about  7  grams  of  potassium  acetate  dissolved  in  a  Uttle  water. 
After  ten  minutes  dilute  with  an  equal  voliune  of  alcohol,  and  allow  to  stand  in  a 
cold  place  for  some  hours.  Filter  off  the  crystalline  product  and  examine  under 
microscope  (see  Fig.  124).  To  remove  the  small  amount  of  potassitun  sulphate 
which  it  contains  stir  up  with  twice  its  weight  of  water,  filter,  wash  with  a  little 
water  and  then  with  alcohol.  The  preparation  should  be  snow  white.  Yield, 
85-90  per  cent. 

Place  the  finely  powdered  recrystallized  creatinine  zinc  chloride  in  a  dry 
flask  and  treat  with  seven  times  its  weight  (by  volume)  of  concentrated  aqueous 
ammonia.  Warm  sUghtly  and  agitate  gently  until  a  clear  solution  is  obtained, 
care  being  taken  to  drive  off  no  more  ammonia  during  the  warming  than  is 
necessary  to  obtain  a  clear  solution.  Stopper  the  flask,  allow  to  cool,  place  in 
the  ice-box  for  an  hour  or  more.  Pure  creatinine  crystaUizes  out.  It  may  be 
recrystallized  from  boiUng  alcohol  or  concentrated  ammonia,  but  this  is  usually 
unnecessary.  The  product  is  perfectly  pure  and  can  be  used  as  a  standard  in 
the  quantitative  determination  of  creatine  and  creatinine.  See  chapters  on 
Quantitative  Analysis  of  Urine  and  Blood. 

I.  Preparation  of  Creatine. — Creatine  may  be  prepared  from  creatinine  zinc 
chloride  by  decomposition  with  calcium  hydrate,  the  process  being  one  of  hydrol- 
ysis (Benedict). 

One  hundred  grams  of  creatinine  zinc  chloride  are  treated  with  about  700  c.c. 
of  water  in  a  large  casserole  and  the  mixture  heated  to  boiling;  15c  grams  of  pure 
powdered  calcium  hydrate  are  then  added,  with  stirring,  and  the  mixture  boiled 
gently  for  20  minutes  (with  occasional  stirring).  The  hot  mixture  is  then  filtered 
with  suction,  the  residue  being  washed  with  hot  water.  The  filtrate  is  then  treated 
with  hydrogen  sulphide  gas  for  a  few  minutes  and  poured  through  a  folded  filter 
to  remove  the  zinc.  The  filtrate  is  acidified  by  the  addition  of  about  5  c.c.  of  glacial 
acetic  acid  and  boiled  down  rapidly  to  a  volume  of  about  200  c.c.  This  solution 
is  allowed  to  stand  over  night,  preferably  in  a  cool  place.  The  next  day  the  crys- 
tallized creatine  is  filtered  off  with  suction,  washed  with  a  very  little  cold  water, 
and  then  thoroughly  washed  with  alcohol  and  dried. ^  This  product  is  then  recrys- 
tallized by  dissolving  in  about  seven  times  its  weight  of  boiling  water  and  allowing 

'  The  filtrate  obtained  at  this  point  should  be  diluted  with  alcohol  and  treated  with  zinc 
chloride  (50  c.c.  of  a  30  per  cent  solution)  for  recovery  of  the  unconverted  creatinine. 


URINE 


38: 


the  solution  to  cool  slowly  and  stand  for  some  hours.  This  product  should  be  per- 
fectly pure  creatine.  If  necessary  it  can  be  recrystallized  with  verj'  little  loss.  The 
crystallized  product  should  be  filtered  off,  washed  with  alcohol  and  ether  and  dried 
in  air  for  about  half  an  hour.  Thus  obtained  the  creatine  contains  water  of  crystal- 
lization which  it  loses  very  readily  upon  exposure  to  air.  To  prepare  creatine  which 
can  be  weighed  with  absolute  exactness  it  is  necessary  to  dehydrate  this  product  by 
heating  for  some  hours  at  about  95°. 

The  yield  in  this  process  is  about  18  grams  of  recrystallized  creatine,  and 
about  55  grams  of  creatinine  zinc  chloride  recovered.  Longer  boiling  with  lime 
does  not  bring  about  a  greater  yield,  as  after  the  20-minute  point  creatine  is 
decomposed  almost  exactly  as  fast  as  it  is  formed. 

Examine  the  crystals  of  creatine  under  the  microscope  and  compare  with  illus- 
tration in  Chapter  XIX  on  Muscular  Tissue.  For  other  creatine  tests  see  Chapter 
XIX. 


F'iG.   124. — Creatinixe-zixc  Chloride.     iSalkowski.) 


3.  Nitro-prusside  Test  (Weyl). — Take  5  c.c.  of  urine  in  a  test-tube,  add  a  few 
drops  of  sodium  nitro-prusside  and  render  the  solution  alkaline  with  potassium 
hydroxide  solution.  A  ruby-red  color  results  which  soon  turns  yellow. 
See  Legal's  test  for  acetone,  page  437. 

4.  Nitro-prusside-acetic  Acid  Test  (Salkowski). — To  the  yellow  solution  ob- 
tained in  Weyl's  test  above  add  an  excess  of  acetic  acid  and  apply  heat.  A  green 
color  results  and  is  in  turn  displaced  by  a  blue  color.  A  precipitate  of  Prussian 
blue  may  form. 

5.  Picric  Acid  Reaction  (Jaffe). — Place  5  c.c.  of  urine  in  a  test-tube,  add  an 
aqueous  solution  of  picric  acid  and  render  the  mixture  alkaline  with  potassium 
hydroxide  solution.  A  red  color  is  produced  which  turns  yellow  if  the  solution  be 
acidified.  Glucose  gives  a  similar  red  color  but  only  upon  the  application  of  heat. 
This  color  reaction  observed  when  creatinine  in  alkaline  solution  is  treated  with 
picric  acid  is  the  basic  principle  of  Folin's  colorimetric  method  for  the  quantitative 
determination  of  creatinine  (see  page  506 j. 

ETHEREAL  SULPHATES 

The  most  important  of  the  ethereal  sulphates  found  in  the 
urine  are  phowl-sulphurk  acid,  p-crcsol-sulpJiuric  acid,  indoxyl-sidphuric 

25 


386  PHYSIOLOGICAL   CHEMISTRY 

acid,  and  skatoxyl-sulphuric  acid.  Pyrocatechol-sulphuric  acid  also 
occurs  in  traces  in  human  urine.  The  total  output  of  ethereal  sulphuric 
acid  (as  SO3)  varies  ordinarily  from  o.i  gram  to  0.25  gram  for  24  hours 
and  comprise  5-15  per  cent  of  the  total  sulphur.  In  health  the  ratio 
of  ethereal  sulphuric  acid  to  inorganic  sulphuric  acid  is  about  i  :  10. 
These  ethereal  sulphuric  acids  originate  in  part  from  the  phenol,  cresol, 
indole  and  skatole  formed  in  the  putrefaction  of  protein  material  in 
the  intestine.  The  phenol  passes  to  the  liver  where  part  of  it  is  conju- 
gated to  form  phenol  potassium  sulphate  and  appears  in  this  form  in  the 
urine  whereas  the  indole  and  skatole  undergo  a  preliminary  oxidation  to 
form  indoxyl  and  skotoxyl  respectively  before  their  conjugation  and 
elimination. 

It  was  formerly  generally  considered  that  each  of  the  ethereal  sul- 
phuric acids  was  formed  principally  in  the  putrefaction  of  protein 
material  in  the  intestine  and  that  therefore  a  determination  of  the  total 
ethereal  sulphuric  acid  content  of  the  urine  was  an  index  of  the  extent  to 
which  these  putrefactive  processes  were  proceeding  within  the  organism. 
Folin,  however,  conducted  a  series  of  experiments  which  seemed  to 
show  that  the  ethereal  sulphuric  acid  content  of  the  urine  did  not  afford 
an  index  of  the  extent  of  intestinal  putrefaction,  since  these  bodies 
arise  only  in  part  from  putrefactive  processes.  He  claims  that  the 
ethereal  sulphuric  acid  excretion  represents  a  form  of  sulphur  metaboHsm 
which  is  more  in  evidence  upon  a  diet  containing  a  very  small  amount  of 
protein  or  upon  a  diet  containing  absolutely  no  protein.  The  ethereal 
sulphuric  acid  content  of  the  urine  diminishes  as  the  total  sulphur  con- 
tent diminishes  but  the  percentage  decrease  is  much  less.  Therefore 
when  considered  from  the  standpoint  of  the  total  sulphuric  acid  content 
the  ethereal  sulphuric  acid  content  is  not  diminished  but  is  increased, 
although  the  total  sulphuric  acid  content  is  diminished.  Folin's  experi- 
ments also  seem  to  show  that  the  indoxyl  sulphuric  acid  (indoxyl  potas- 
sium sulphate  or  indican)  content  of  the  urine  does  not  originate  to  any 
degree  from  the  metabolism  of  protein  material  but  that  it  arises  in 
great  part  from  intestinal  putrefaction  and  that  the  excretion  of  indoxyl 
sulphuric  acid  may  alone  be  taken  as  a  rough  index  of  the  extent  of  putre- 
factive processes  within  the  intestine.     Indoxyl  sulphuric  acid, 

CH 

HC      C       C(0.S03H), 

!     II      II 

HC      C      CH 

\/\/ 
CH  NH 


URINE  387 

therefore,  which  occurs  in  the  urine  as  indoxyl  potassium  sulphate  or 

indican, 

CH 

^\ 
HC      C— CCO.SOsK), 

I       II       II 
HC      C      CH 

CH  NH 

is  clinically  the  most  important  of  the  ethereal  sulphuric  acids.  Under 
normal  conditions  from  4  to  20  mg.  of  indican  are  excreted  per  day. 
The  variations  are  due  mainly  to  diet,  a  high  meat  diet  causing  an 
increase  and  a  carbohydrate  diet  a  decrease.  Pathologically  the  great- 
est increases  are  found  in  disorders  involving  increased  putrefaction  and 
stagnation  of  intestinal  contents.  Bacterial  decomposition  of  body 
protein  as  in  gangrene,  putrid  pus  formation,  etc.,  gives  rise  to  an 
increased  indican  excretion. 

It  was  formerly  believed  that  the  phenol  was  excreted  practically 
quantitatively  in  the  conjugated  form'.  Recent  work  of  Folin  and  Denis^ 
seems  to  indicate  that  this  is  not  true.  Only  pari  of  the  phenols  formed 
in  intestinal  putrefaction  are  excreted  in  the  conjugated  form,  the 
remainder  being  excreted  as  free  phenol.  The  phenol  output  tends  to 
vary  directly  but  not  proportionally  with  the  protein  ingestion.  The 
total  phenol  excretion  of  normal  men  on  an  ordinary  mixed  diet  averages 
around  0.4  gram  per  day. 

Tests  for  Indican^ 

I.  Jaffa's  Test. — Nearly  fill  a  test-tube  with  a  mixture  composed  of  equal 
volimies  of  concentrated  HCl  and  the  xirine  vmder  examination.  Add  2-3  c.c. 
of  chloroform  and  a  few  drops  of  a  calcium  hypochlorite  solution,  place  the  thumb 
over  the  end  of  the  test-tube  and  shake  the  tube  and  contents  thoroughly. 
The  chloroform  is  colored  more  or  less,  according  to  the  amount  of  indican  pres- 
ent. Ordinarily  a  blue  color  due  to  the  formation  of  indigo-blue  is  produced ; 
less  frequently  a  red  color  due  to  indigo-red  may  be  noted. 

Repeat]  this  test  on  some  of  this  same  mine  to  which  formaldehyde  has 
been  added.  Is  there  any  variation  in  the  reaction  from  what  you  previously 
obtained? 

This  is  the  reaction  (see  also  pages  212-213): 

'  Folin  and  Denis:  Jour.  Biol.  Chem.,  22,  309,  1915. 

'  The  urine  should  always  be  examined  fresh  if  this  is  possible.  In  any  event  formalde- 
hyde should  never  be  used  as  a  preservative  for  such  urines  as  are  to  be  e.xamined  for  indican 
by  means  of  any  test  involving  hypochlorite  or  potassium  permanganate.  The  formalde- 
hyde through  its  reducing  power  lowers  the  oxidizing  efliciency  of  the  mixture.  The  forma- 
tion of  formic  acid  from  the  aldehyde  may  also  interfere. 


388  PHYSIOLOGICAL    CHEMISTRY 

CH 


HC      C — C.OH 

2     I     !|     II      +  20- 

HC      C      CH 


CH  NH 

Indoxyt,  CsHtXO. 

CH  CH 

/\  /% 

HC      C      CO     O.C— C      CH 

I       II        I  I       II        I      +  2H2O 

HC      C      c C     C      CH 


CH  NH  NH  CH 

Indigo-blue,  CiiHioX202. 

2.  Obennayer's  Test. — Nearly  fill  a  test-tube  with  a  mixture  composed  of 
equal  volumes  of  Obennayer's  reagent^  and  the  urine  under  examination. 
Add  2-3  c.c.  of  chloroform,  place  the  thumb  over  the  end  of  the  test-tube  and 
shake  thoroughly.     How  does  this  compare  with  Jaffe's  test? 

3.  Jolles'  Reaction.- — To  10  c.c.  of  urine  add  i  c.c.  of  a  5  per  cent  alcoholic 
thymol  solution  and  shake.  Add  about  10  c.c.  of  fuming  HCl  containing  5 
grams  of  ferric  chloride  per  hter.  Shake  again  carefully  and  let  stand  for  15 
minutes.  Add  about  4  c.c.  of  chloroform  and  extract  the  pigment  by  repeated 
gentle  shaking.  The  chloroform  becomes  intensely  violet.  0.0032  mg.  of  indi- 
can  can  be  detected  in  10  c.c.  of  urine.  It  is  much  the  most  delicate  test  for 
indican. 

CO.NH.CH2.COOH. 

/\ 
HIPPURIC  ACID, 


This  acid  occurs  normally  in  the  urine  of  both  the  carnivora  and 
herbivora  but  is  much  more  abundant  in  the  urine  of  the  latter.  It  is 
formed  by  a  synthesis  of  benzoic  acid  and  glycocoll  which  takes  place 
in  the  kidneys  and  elsewhere^.  The  glycocoll  comes  from  decomposi- 
tion of  protein.  The  benzoic  acid  thus  utilized  may  come  from  (i)  pre- 
formed benzoic  acid  of  fruits  and  vegetables;  (2)  other  aromatic  com- 
pounds of  fruits  and  vegetables;  (3)  aromatic  amino-acids  (tyrosine 
and  phenylalanine)  from  the  alimentary  tract.  The  average  excretion 
of  hippuric  acid  by  an  adult  man  for  24  hours  under  normal  conditions 
is  about  0.7  gram.  Hippuric  acid  crystallizes  in  needles  or  rhombic 
prisms  (see  Fig.  125,  p.  389)  the  particular  form  depending  upon  the 

^  Obermayer's  reagent  is  prepared  by  adding  2-4  grams  of  ferric  chloride  to  a  liter  of  con- 
centrated HCl  (sp.  gr.  1. 19). 

"^  Zeit.  physiol.  Chem.,  94,  79,  1915. 

*  Kingsbury  and  Bell:  Jour.  Biol.  Chem.,  21,  297,  1915. 


URINE 


389 


rapidity  of  crystallization.  Pure  hippuric  acid  melts  at  iSy^C.  The 
most  satisfactory  method  for  the  isolation  of  hippuric  acid  from  the 
urine  in  crystalline  [form  is  that  proposed  by  Roaf  (see  below).  It 
is  easily  soluble  in  alcohol  or  hot  water,  and-  only  slightly  soluble  in 
ether.  The  output  of  hippuric  acid  is  increased  in  diabetes  owing  prob- 
ably to  the  ingestion  of  much  protein  and  fruit.  Plums,  prunes  and 
cranberries  in  particular  increase  the  hippuric  acid  output  considerably 
due  to  their  relatively  high  content  of  benzoic  acid.  Hippuric  acid 
is  decreased  in'fevers  and  in  certain  kidney  disorders  where  the  synthetic 


Fig.   i2v — Hippuric  Acid. 


activity  of  the  renal  cells  is  diminished.     It  may  be  determined  quan- 
titatively by  methods  given  in  Chapter  XXVI. 

Experiments  on  Hippuric  Acid 

I.  Separation  from  the  Urine. — (a)  First  Method.  Render  500-1000  c.c. 
of  urine  of  the  horse  or  cow'  alkahne  with  milk  of  lime,  boil  for  a  few  moments  and 
filter  while  hot.  Concentrate  the  filtrate,  over  a  burner,  to  a  small  volume.  Cool 
the  solution,  acidify  it  strongly  with  concentrated  hydrochloric  acid  and  stand  it 
in  a  cool  place  for  24  hours.  Filter  off  the  crystals  of  hippuric  acid  which  have 
formed  and  wash  them  with  a  little  cold  water.  Remove  the  crystals  from  the 
paper,  dissolve  them  in  a  very  small  amount  of  hot  water  and  percolate  the  hot 
solution  through  thoroughly  washed  animal  charcoal,  being  careful  to  wash  out 
the  last  portion  of  the  hippuric  acid  solution  with  hot  water.     Filter,  concentrate 

'  If  urine  of  the  horse  or  cow  is  not  available  human  urine  may  serve  the  purpose  fuUj- 
as  well  provided  means  are  taken  to  increase  its  content  of  hippuric  acid.  This  may  be  con- 
veniently accomplished  by  ingesting  2  prams  of  ammonium  benzoate  at  night.  (See 
chapter  on  Metabolism).  The  fraction  of  urine  passed  in  the  morning  will  be  found  to  have 
a  high  content  of  hippuric  acid.  The  ammonium  benzoate  is  in  no  way  harmful.  In  case 
ammonium  benzoate  is  not  available  sodium  benzoate  may  be  substituted. 


390  PHYSIOLOGICAL   CHEMISTRY 

the  filtrate  to  a  small  volume  and  stand  it  aside  for  crystallization.  Examine  the 
crystals  imder  the  microscope  and  compare  them  with  those  in  Fig.  125,  page  389. 
This  method  is  not  as  satisfactory  as  Roaf's  method  (see  below). 

(b)  Roaf's  Method. — ^Place  500  c.c.  of  urine  of  the  horse  or  cow  in  a  cas- 
serole or  precipitating  jar  and  add  an  equal  volume  of  a  saturated  solution  of 
ammonium  sulphate^  and  7.5  c.c.  of  concentrated  sulphuric  acid.  Permit  the 
mixture  to  stand  for  24  hours  and  remove  the  crystals  of  hippuric  acid  by  filtration. 
Purify  the  crystals  by  recrystaUization  according  to  the  directions  given  above 
imder  First  Method.  Examine  the  crystals  imder  the  microscope  and  compare 
them  with  those  given  in  Fig.  125,  page  389. 

If  suflficient  urine  is  not  available  to  permit  the  use  of  500  c.c.  a  smaller  volume 
may  be  used  inasmuch  as  it  is  possible,  by  the  above  technic,  to  isolate  hippuric 
acid  in  crystalline  form  from  as  small  a  volume  as  25-50  c.c.  of  herbivorous  urine. 
The  greater  the  amoimt  of  ammoniimi  sulphate  added  the  more  rapid  the  crystal- 
Uzation  imtil  at  the  saturation  point  the  crystals  of  hippuric  acid  sometimes  form  in 
about  ten  minutes. 

2.  Melting-point. — ^Determine  the  melting-point  of  the  hippuric  acid  pre- 
pared in  the  above  experiment  (see  page  375). 

3.  SolubiUty. — ^Test  the  solubiUty  of  hippuric  acid  in  hot  and  cold  water  and 
in  alcohol,  and  ether. 

4.  Formation  of  Nitro-Benzene  (Liicke's  Reaction). — To  a  little  hippuric  acid 
in  a  small  porcelain  dish  add  1-2  c.c.  of  concentrated  HNO3  and  evaporate  to  dry- 
ness on  a  water-bath.  Transfer  the  residue  to  a  dry  test-tube,  apply  heat,  and 
note  the  odor  of  the  artificial  oil  of  bitter  almonds  (nitrobenzene). 

5.  Spiro's  Reaction. 2 — Warm  the  hippuric  acid  with  acetic  anhydride,  anhy- 
drous sodium  acetate  and  benzaldehyde.  After  one-half  hour  permit  the  solution 
to  cool.  Note  the  formation  of  crystals  of  the  lactimide  of  phenylaminocinnamic 
acid,  melting-point  165-166°.  Heat  some  of  the  crystals  with  concentrated  sodium 
hydroxide  until  ammonia  is  given  off.  Acidify  and  note  the  formation  of  phenyl- 
pyroracemic  acid  (C6H6CH2.CO.COOH).     This  acid  is  soluble  in  ether. 

6.  Sublimation. — ^Place  a  few  crystals  of  hippuric  acid  in  a  dry  test-tube  and 
apply  heat.  The  crystals  are  reduced  to  an  oily  fluid  which  soUdifies  in  a  crystal- 
Une  mass  upon  cooling.  When  stronger  heat  is  appUed  the  Uquid  assumes  a 
red  color  and  finally  yields  a  sublimate  of  benzoic  acid  and  the  odor  of  hydro- 
cyanic acid. 

7.  Formation  of  Ferric  Salt. — ^Render  a  small  amount  of  a  solution  of  hip- 
puric acid  neutral  with  dilute  potassium  hydroxide.  Now  add  1-3  drops  of 
neutral  ferric  chloride  solution  and  note  the  formation  of  the  ferric  salt  of  hip- 
puric acid  as  a  cream-colored  precipitate. 

8.  Synthesis  of  Hippuric  Acid. — To  some  of  the  glycocoU  prepared  in  the  last 
experiment  or  furnished  by  the  instructor,  add  a  little  water,  about  i  c.c.  of  benzoyl 
chloride  and  render  alkaline  with  potassium  hydroxide  solution.  Stopper  the  tube 
and  shake  it  until  no  more  heat  is  evolved.  Now  render  strongly  alkaline  with 
potassium  hydroxide  and  shake  the  mixture  until  no  odor  of  benzoyl  chloride  can  be 
detected.  Cool,  acidify  with  hydrochloric  acid,  add  an  equal  volume  of  petroleum 
ether,  and  shake  thoroughly  to  remove  the  benzoic  acid.  (Evaporate  this  solution 
and  note  the  crystals  of  benzoic  acid.  Compare  them  with  those  shown  in  Fig.  127, 
page  396.)     Decant  the  ethereal  solution  into  a  porcelain  dish  and  extract  again 

1 125  grams  of  solid  ammonium  sulphate  may  be  substituted. 
^  Spiro:  ZeiL  physiol.  Chem.,  28,  174,  1899. 


URINE  391 

with  ether.  The  hippuric  acid  remains  in  the  aqueous  solution.  Filter  it  off  and 
wash  it  with  a  small  amount  of  cold  water  while  still  on  the  filter.  Remove  it  to 
a  small,  shallow  vessel,  dissolve  it  in  a  small  amount  of  hot  water  and  set  it  aside 
for  crystallization.  Examine  the  crystals  microscopically  and  compare  them  with 
those  in  Fig.  125,  page  389. 

The  chemistry  of  the  synthesis  is  represented  thus: 

CH2-NH2  COCl  0C-NH-CH2-C00H. 

COOH  \/  \/ 

Glycocoll.  Benzoyl  chloride.        Hippuric  acid. 


OXALIC  ACID, 


COOH 
COOH 


Oxalic  acid  is  a  constituent  of  normal  urine,  about  15-20  mg.  being 
eliminated  in  24  hours.  It  is  present  in  the  urine  as  calcium  oxalate 
which  is  kept  in  solution  through  the  medium  of  the  acid  phosphates. 
The  origin  of  the  oxalic  acid  content  of  the  urine  is  not  well  under- 
stood. It  is  eliminated,  at  least  in  part,  unchanged  when  ingested,  there- 
fore since  many  of  the  common  articles  of  diet,  e.g.,  asparagus,  apples, 
cabbage,  grapes,  lettuce,  spinach,  tomatoes,  etc.,  contain  oxalic  acid 
(oxalates)  it  seems  probable  that  the  ingested  food  supplies  a  portion  of 
the  oxalic  acid  found  in  the  urine.  There  is  also  experimental  evidence 
that  part  of  the  oxalic  acid  of  the  urine  is  formed  within  the  organism 
in  the  course  of  protein  and  fat  metabolism.  It  has  also  been  suggested 
that  oxalic  acid  may  arise  from  an  incomplete  combustion  of  carbo- 
hydrates, especially  under  certain  abnormal  conditions.  Patholog- 
ically, oxalic  acid  is  found  to  be  increased  in  amount  in  diabetes  mellitus, 
in  organic  diseases  of  the  liver,  and  in  various  other  conditions  which  are 
accompanied  by  a  derangement  of  the  oxidation  mechanism.  An 
abnormal  increase  of  oxalic  acid  is  termed  oxaluria.  A  considerable 
increase  in  the  content  of  oxalic  acid  may  be  noted  unaccompanied  by 
any  other  apparent  symptom.  Calcium  oxalate  crystallizes  in  at  least 
two  distinct  forms,  dumb-bells  and  octahedra  (Fig.  134,  page  459). 

Experiments 

Preparation  of  Calcium  Oxalate. — First  Method. — Place  200-250  c.c.  of 
urine  in  a  beaker,  add  5  c.c.  of  a  saturated  solution  of  calcium  chloride,  make  the 
urine  slightly  acid  with  acetic  acid,  and  stand  the  beaker  aside  in  a  cool  place  for 
24  hours.  Examine  the  sediment  under  the  microscope  and  compare  the  crystal- 
line forms  with  those  shown  in  Fig.  134,  page  459. 

Second  Method. — Proceed  as  above,  replacing  the  acetic  acid  by  an  excess  of 
ammonium  hydroxide  and  filtering  off  the  precipitate  of  phosphates. 


392 


PHYSIOLOGICAL   CHEMISTRY 


NEUTRAL  SULPHUR  COMPOUNDS 

Under  this  head  may  be  classed  such  bodies  as  cystine  (see  page  75), 
chondroitin-sulphuric  acid,  oxyproteic  acid,  alloxyproteic  acid,  uroferric 
acid,  methyl  mercaptan,  ethyl  sulphide,  thiocyanates  and  taurine 
derivatives.  The  sulphur  content  of  the  bodies  just  enumerated  is 
generally  termed  unoxidized  or  neutral  sulphur  in  order  that  it  may  not 
be  confused  with  the  acid  or  oxidized  sulphur  which  occurs  in  the 
inorganic  sulphuric  acid  and  ethereal  sulphuric  acid  forms.  Ordinarily 
the  neutral  sulphur  content  of  normal  human  urine  is  5-25  per  cent 
of  the  total  sulphur  content  (see  "Sulphur  Partition"  in  chapter  on 
Metabolism.)     The   actual  amount  excreted  may  be  0.2-0.4  grams 


Fig.  126. — Allantoin,  from  Cat's  Urine. 

a  and  6,  Forms  in  which  it  crystallized  from  the  urine;  c,  recrystallized  allantoin.     (Drawn 
from  micro-photographs  furnished  by  Prof.  Lafayette  B.  Mendel  of  Yale  University.) 

per  day,  calculated  as  SO3.  Its  origin  is  mainly  endogenous.  The 
excretion  is  fairly  constant  for  any  given  individual  in  spite  of  dietary 
changes.  In  tuberculosis,  cancer,  cystinuria,  etc.,  the  amount  may 
be  relatively  or  absolutely  increased. 

NH.CH.NH 


ALLANTOIN,  OC  CO 

I         !         i 

NH.CO  NH2 

Allantoin  is  found  in  the  urine  of  practically  all  mammals  including 
man.  In  human  urine  it  occurs  in  very  small  amount  (5-15  mg.  per 
day)  whereas  in  the  case  of  all  other  mammals  investigated  except 
anthropoid  apes,  it  is  the  principal  end-product  of  purine  metabolism 


URINE  393 

and  may  constitute  90  per  cent  or  over  of  the  total  purine  output.^ 
Allantoin  is  formed  by  the  oxidation  of  uric  acid  and  the  output  is 
increased  by  the  feeding  of  thymus  or  pancreas  to  lower  animals. 
When  pure  it  crystallizes  in  prisms  (Fig.  126,  page  392)  and  when  impure 
in  granules  and  knobs.  Pathologically,  it  has  been  found  increased  in 
diabetes  insipidus  and  in  hysteria  with  convulsions  (Pouchet).  Mendel 
and  Dakin-  have  shown  that  allantoin  is  optically  inactive  notwith- 
standing the  fact  that  it  contains  an  asymmetric  carbon  atom.  This 
phenomenon  they  believe  to  be  due  to  tautomeric  change.  Wiechowski 
has  suggested  an  excellent  method  for  the  quantitative  determination 
of  allantoin.     (See  Chapter  XXVI.) 

Experiments 

1.  Separation  from  the  Urine. ^ — Meissner's  Method. — Precipitate  the  urine 
with  baryta  water.  NeutraUze  the  filtrate  carefully  with  dilute  sulphuric  acid, 
filter  immediately,  and  evaporate  the  filtrate  to  incipient  crystallization.  Com- 
pletely precipitate  this  warm  fluid  with  95  per  cent  alcohol  (reserve  the  precipi- 
tate). Decant  or  filter  and  precipitate  the  solution  by  ether.  Combine  the 
ether  and  alcohol  precipitates  and  extract  with  cold  water  or  hot  alcohol;  allan- 
toin remains  undissolved.  Bring  the  allantoin  into  solution  in  hot  water  and 
recrystallize. 

2.  Preparation  from  Uric  Acid. — Dissolve  4  grams  of  uric  acid  in  100  c.c.  of 
water  rendered  alkaUne  with  potassium  hydroxide.  Cool  and  carefully  add  3 
grams  of  potassium  permanganate.  Filter,  immediately  acidulate  the  filtrate 
with  acetic  acid  and  allow  it  to  stand  in  a  cool  place  over  night.  Filter  off  the 
crystals  and  wash  them  with  water.  Save  the  wash  water  and  filtrate,  unite 
them  and  after  concentrating  to  a  small  volume  stand  away  for  crystaUization. 
Now  combine  all  the  crystals  and  recrystallize  them  from  hot  water.  Use  these 
crystals  in  the  experiments  which  follow. 

3.  Microscopical  Examination. — Examine  the  crystals  made  in  the  last 
experiment  and  compare  them  with  those  shown  in  Fig.  126. 

4.  Solubility. — Test  the  solubility  of  allantoin  in  cold  and  hot  water,  cold  and 
hot  alcohol  and  in  ether. 

5.  Reaction. — Dissolve  a  crystal  in  water  and  test  the  reaction  to  htmus. 

6.  Furfural  Test  (Schiff ). — Place  a  few  crystals  of  allantoin  on  a  test-tablet  or 
in  a  porcelain  dish  and  add  1-2  drops  of  a  concentrated  aqueous  solution  of  fur- 
fural and  1-2  drops  of  concentrated  hydrochloric  acid.  Observe  the  formation  of 
a  yellow  color  which  turns  to  a  hght  purple  if  allowed  to  stand.  This  test  is  given 
by  urea  but  not  by  uric  acid. 

7.  Murexide  Test. — Try  this  test  according  to  the  directions  given  on  page 
380.     Note  that  allantoin  fails  to  respond. 

8.  Reduction  of  Fehling's  Solution.  Make  this  test  in  the  usual  way  isee 
page  416)  except  that  the  boiling  must  be  prolonged  and  excessive.     Ultimately 

•  Wiechowski:  "Die  Purinstoffe  und  das  .Mlantoin"  in  Xeul)auiT  and  Huppert's  "Ana- 
lyse des  Harris,'^  Wiesbaden,  1913. 

*  Mendel  and  Dakin:  Jour.  Bwl.  Clicm.,  7,  153,  iqio. 

'The  urine  of  the  dog  after  thymus,  pancreas,  or  uric  acid  feeding  may  be  employed. 


394  PEEYSIOLOGICAL   CHEMISTRY 

the  allantoin  will  reduce  the  solution.    Compare  with  the  result  on  uric  acid, 
page  381. 

AMINO-ACIDS' 

Certain  of  these  acids  are  always  present  in  normal  urine.  The 
excretion  of  total  amino-acid  nitrogen  by  a  normal  adult  averages 
0.4-1.0  gram  per  day  or  about  2-6  per  cent  of  the  total  nitrogen. 
Free  amino-acid  nitrogen  (see  van  Slyke  procedure,  Chapter  IV)  is 
considerably  less  than  this,  and  ordinarily  constitutes  0.5-1  per  cent 
of  the  total  nitrogen.  The  amount  may  be  largely  increased  in  disorders 
associated  with  tissue  waste,  e.g.,  typhoid,  acidosis,  pronounced  atrophy 
of  the  liver,  etc.     For  tests  on  amino-acids  see  Chapter  IV. 

AROMATIC  OXYACIDS 

Two  of  the  most  important  of  the  oxyacids  are  parahydroxy- 
phenyl-acetic  acid, 

CH2.COOH, 


OH 

and  par ahydroxy- phenyl-propionic  acid, 

CH2.CH2.COOH. 


OH 

They  are  products  of  the  putrefaction  of  protein  material  and  tyrosine 
is  an  intermediate  stage  in  their  formation.  Both  these  acids  for  the 
most  part  pass  unchanged  into  the  urine  where  they  occur  normally  in 
very  small  amount.  The  content  may  be  increased  in  the  same  manner 
as  the  phenol  content,  in  particular  by  acute  phosphorus  poisoning.  A 
fraction  of  the  total  aromatic  oxyacid  content  of  the  urine  is  in  combina- 
tion with  sulphuric  acid,  but  the  greater  part  is  present  in  the  form 
of  salts  of  sodium  and  potassium. 

Homogentisic  Acid  or  di-hydroxyphenyl-acetic  acid, 

OH 

ICH2.COOH, 

OH 

*  For  a  full  discussion  see  Underhill's  "The  Physiology  of  the  Amino  Acids,"  Yale 
University  Press,  November,  1915. 


URINE  395 

is  another  important  oxyacid  sometimes  present  in  the  urine.  Under 
the  name  glycosuric  acid  it  was  first  isolated  from  the  urine  by  Prof. 
John  ^Marshall  of  the  University  of  Pennsylvania;  subsequently  Bau- 
raann  isolated  it  and  determined  its  chemical  constitution.  It  occurs  in 
cases  of  alcaptonuria.  A  urine  containing  this  oxyacid  turns  greenish- 
brown  from  the  surface  downward  when  treated  with  a  little  sodium 
hydroxide  or  ammonia.  If  the  solution  be  stirred  the  color  very  soon 
becomes  dark  brown  or  even  black.  Homogentisic  acid  reduces 
alkaline  copper  solutions  but  not  alkaline  bismuth  solutions.  Uro- 
leucic  acid  is  similar  in  its  reactions  to  homogentisic  acid. 
Hydroxymandelic  Acid  or  parahydroxyphenyl-glycolic  acid, 

OH 


CH(OH).COOH, 

has  been  detected  in  the  urine  in  cases  of  yellow  atrophy  of  the  liver. 
Kynurenic  Acid  or  7-oxy-/3-quinoline  carbonic  acid, 

CH  COH 
HC      C      C.COOH, 

I     I!      I 

HC      C      CH 

\/\^ 
CH  N 

is  present  in  the  urine  of  the  dog  and  has  recently  been  detected  by 
Swain  in  the  urine  of  the  coyote.  To  isolate  it  from  the  urine  proceed 
as  follows:  Acidify  the  urine  with  hydrochloric  acid  in  the  proportion 
1:25.  From  this  acid  fluid  both  the  uric  acid  and  the  kynurenic  acid 
separate  in  the  course  of  24-48  hours.  Filter  off  the  combined  crys- 
talline deposit  of  the  two  acids,  dissolve  the  kynurenic  acid  in  dilute 
ammonia  (uric  acid  is  insoluble),  and  reprecipitate  it  with  hydrochloric 
add.  If  a  solution  containing  kynurenic  acid  be  evaporated  to  dryness 
with  hydrochloric  acid  and  potassium  chlorate,  a  reddish  residue  is 
obtained  which  becomes  first  brownish  green  and  then  emerald  green  on 
adding  ammonia  (Jaffe). 

Kynurenic  acid  may  be  quantitatively   determined  by  Capaldi's 
method.^ 

COOH. 


BENZOIC  ACID, 

\/ 


Zeilschrifi  fiir  physiologische  Chemie,  23,  92,  1897. 


39^ 


PHYSIOLOGICAL   CHEMISTRY 


Benzoic  acid  has  been  detected  in  the  urine  of  the  rabbit  and  dog.  It  is 
also  said  to  occur  in  human  urine  accompanying  renal  disorders.  The 
benzoic  acid  probably  originates  from  a  fermentative  decomposition  of 
the  hippuric  acid  of  the  urine.  Benzoic  acid  and  glycocoll  are  synthe- 
sized in  the  kidney  and  elsewhere^  to  form  hippuric  acid  (see  page  585). 
Certain  fruits  and  berries  contain  considerable  benzoic  acid;  e.g.,  cran- 
berries have  been  shown  to  contain  0.06  per  cent." 

Experiments 

1.  Solubility. — Test  the  solubility  of  benzoic  acid  in  water,  alcohol,  and  ether. 

2.  Crystalline  Form. — Recrystallize  some  benzoic  acid  from  hot  water,  ex- 
amine the  crystals  under  the  microscope,  and  compare  them  with  those  re- 
produced in  Fig.  127. 


Fig.   127. — Benzoic  Acid. 

3.  Sublimation. — Place  a  little  benzoic  acid  in  a  test-tube  and  heat  over  a 
flame.  Note  the  odor  which  is  evolved  and  observe  that  the  acid  sublimes  in  the 
form  of  needles. 

4.  Dissolve  a  little  sodium  benzoate  in  water  and  add  a  solution  of  neutral 
ferric  chloride.  Note  the  production  of  a  brownish -yellow  precipitate  (salicylic 
acid  gives  a  reddish -violet  color  under  the  same  conditions).  Add  ammoniimi 
hydroxide  to  some  of  the  precipitate.  It  dissolves  and  ferric  hydroxide  is  formed. 
Add  a  Uttle  hydrochloric  acid  to  another  portion  of  the  original  precipitate  and 
stand  the  vessel  away  over  night.     What  do  you  observe? 


NUCLEOPROTEIN 

The  nubecula  of  normal  urine  has  been  shown  by  one  investigator 
to  consist  of  a  mucoid  containing  12.7  per  cent  of  nitrogen  and  2.3 
per  cent  of  sulphur.     This  body  evidently  originates  in  the  urinary 

'  Kingsbury  .and  Bell:  Jour.  Biol.  Cheni.,  21,  297,  1915. 

-  Radin  [quoted  by  Blatherwick  {Arch.  Int.  Med.,  14,  409,  1914)  from  unpublished  data]. 


URINE  ,397 

passages.  It  is  probably  slightly  soluble  in  the  urine.  Some  investiga- 
tors believe  that  the  body  forming  the  nubecula  of  normal  urine  is 
nucleoprotein  and  not  a  mucin  or  mucoid  as  stated  above.  A  discussion 
of  nucleoprotein  and  related  bodies  occurring  in  the  urine  under  patho- 
logical conditions  will  be  found  on  page  428. 

NH— CO 

OXALURIC  ACID,  CO        | 

NHo    COOH. 

Oxaluric  acid  is  not  a  constant  constituent  of  normal  human  urine, 
and  when  found  occurs  only  in  traces  as  the  ammonium  salt.  Upon 
boiling  oxaluric  acid  it  splits  into  oxalic  acid  and  urea. 

GLUCOSE 

This  sugar  occurs  in  traces  in  normal  urine.  It  is,  however,  not 
present  in  sufficient  concentration  to  be  detected  by  any  of  the  ordinary 
tests  used  in  urine  analysis.  In  certain  pathological  conditions  (pp. 
413  and  523)  the  sugar  in  the  urine  is  notably  increased.  Folin  has 
recently  modified  Benedict's  sugar  test  (see  Chapter  XXIII)  so  it  may 
be  used  to  demonstrate  the  sugar  content  of  normal  urine.  ^ 

ENZYMES 

Various  types  of  enzymes  produced  within  the  organism  are  excreted 
in  both  the  feces  and  the  urine.  In  this  connection  it  is  interesting  to 
note  that  pepsin,  rennin,  lipase  and  an  amylase  have  been  positively 
identified  in  the  urine.  The  occurrence  of  trypsin  in  the  urine,  at  least 
under  normal  conditions,  is  questioned. 

VOLATILE  FATTY  ACIDS 

Acetic,  butyric,  and  formic  acids  have  been  found  under  normal 
conditions  in  the  urine  of  man  and  of  certain  carnivora  as  well  as  in  the 
urine  of  herbivora.  Normally  they  arise  principally  from  the  fermenta- 
tion of  carbohydrates  and  the  putrefaction  of  proteins.  The  acids  con- 
taining the  fewest  carbon  atoms  (formic  and  acetic)  are  found  to  be 
present  in  larger  percentage  than  those  which  contain  a  larger  number  of 
such  atoms.  The  volatile  fatty  acids  occur  in  normal  urine  in  traces, 
the  total  output  for  24  hours  according  to  older  investigators  varying 
from  0.008  gram  to  0.05  gram. 

Pathologically,  the  excretion  of  volatile  fatty  acids  is  increased  in 

^  Folin:  Jour.  Biol.  Client.,  22,  327,  1915. 


398  PHYSIOLOGICAL   CHEMISTRY 

diabetes,  fevers,  and  in  certain  hepatic  diseases  in  which  the  parenchyma 
of  the  liver  is  seriously  affected.  Under  other  pathological  conditions 
the  output  may  be  diminished.  These  variations,  however,  in  the 
excretion  of  the  volatile  fatty  acids  possess  very  little  diagnostic 
value. 

CH3 

PARALACTIC  ACID,  CH(OH) 

COOH. 

Paralactic  acid  is  supposed  to  pass  into  the  urine  when  the  supply 

of  oxygen  in  the  organism  is  diminished  through  any  cause,  e.g.,  in 

eclampsia,  acute  yellow  atrophy  of  the  liver,  carbon-monoxide  poisoning, 

acute  phosphorus  poisoning,  or  epileptic  attacks.     This  acid  has  also 

been  found  in  the  urine  of  healthy  persons  following  the  physical  exercise 

incident  to  prolonged  marching.     Paralactic  acid  has  been  detected  in 

the  urine  of  birds  after  the  removal  of  the  liver.     Underhill  reports  the 

occurrence  of  this  acid  in  the  urine  of  a  case  of  pernicious  vomiting  of 

pregnancy. 

CH2.CO.NH.CH2.COOH. 

/\ 
PHENACETURIC  ACID,  I 

\/ 

Phenaceturic  acid  occurs  principally  in  the  urine  of  herbivorous 
animals.  It  may  be  isolated  from  the  urine  of  the  dog  after  feeding 
phenylacetic  acid. ^  It  is  produced  in  the  organism  through  the  synthesis 
of  glycocoll  and  phenylacetic  acid.  It  is  doubtful  if  it  occurs  in  normal 
human  urine  even  after  the  ingestion  of  phenylacetic  acid.  It  may  be 
decomposed  into  its  component  parts  by  boiling  with  dilute  mineral 
acids.  The  crystalline  form  of  phenaceturic  acid  (small  rhombic  plates 
with  rounded  angles)  resembles  one  form  of  uric  acid  crystal. 

HC  =  C-CH  =  CHCOOH 
UROCANIC  ACID,  HN      N 

CH 

This  acid  has  been  found  in  the  urine  of  dogs,  but  not  in  human 
urine.  It  is  imidazolyl-acrylic  acid.  Hunter^  found  it  among  the 
pancreatic  digestion  products  of  casein.  It  crystallizes  as  sickle-shaped 
crystals. 

1  Sherwin:  Dissertation,  Tubingen,  1915. 
*  Hunter:  Jour.  Biol.  Chetn.,  11,  537,  1913. 


URINE  399 

PHOSPHORIZED  COMPOUNDS 

Phosphorus  in  organic  combination  has  been  found  in  the  urine 
in  such  bodies  as  glycerophosphoric  acid,  which  may  arise  from  the 
decomposition  of  lecithin,  and  phosphocarnic  acid.  It  is  claimed  that 
on  the  average  about  2.5  per  cent  of  the  total  phosphorus  elimination 
is  in  organic  combination. 

PIGMENTS 

There  are  at  least  three  pigments  normally  present  in  human  urine. 
These  pigments  are  urochrome,  urobilin,  and  uroerythrin. 

A.  UROCHROME 

This  is  the  principal  pigment  of  normal  urine  and  imparts  the  char- 
acteristic yellow  color  to  that  fluid.  It  is  apparently  closely  related  to 
its  associated  pigment  urobilin  since  the  latter  may  be  readily  converted 
into  urochrome  through  evaporation  of  its  aqueous-ether  solution.  Uro- 
chrome may  be  obtained  in  the  form  of  a  brown,  amorphous  powder 
which  is  readily  soluble  in  water  and  95  per  cent  alcohol.  It  is  less 
soluble  in  absolute  alcohol,  acetone,  amyl  alcohol  and  acetic  ether,  and 
insoluble  in  benzene,  chloroform,  and  ether.  Urochrome  is  said  to  be  a 
nitrogenous  body  (4.2  per  cent  nitrogen),  free  from  iron.  Urochrome 
is  believed  to  be  identical  with  the  yellow  pigment,  lactocJirome,  of 
milk  whey.^  The  chromogen  of  urochrome,  i.e.,  urochromogen  is  present 
in  the  urine  in  pulmonary  tuberculosis.  Its  presence  is  said  to  be  of 
prognostic  value  (see  page  453). 

B.  UROBILIN 

Urobilin,  which  was  at  one  time  considered  to  be  the  principal  pig- 
ment of  urine,  in  reality  contributes  little  toward  the  pigmentation  of 
this  fluid.  It  is  claimed  that  no  urobilin  is  present  in  freshly  voided  nor- 
mal urine  but  that  its  precursor,  a  chromogen  called  urobilinogen,  is 
present  and  gives  rise  to  urobilin  upon  decomposition  through  the  in- 
fluence of  light.  It  is  claimed  by  some  investigators  that  there  are 
various  forms  of  urobilin,  e.g.,  normal,  febrile,  physiological,  and  patho- 
logical. Urobilin  is  said  to  be  very  similar  to,  if  not  absolutely  identical 
with,  hydrobilirubin  (see  page  222). 

Urobilin  may  be  obtained  as  an  amorphous  powder  which  varies 
in  color  from  brown  to  reddish-brown,  red  and  reddish-yellow,  depend- 
ing upon  the  way  in  which  it  is  prepared.  It  is  easily  soluble  in  ethyl 
alcohol,  amyl  alcohol,  and  chloroform,  and  slightly  soluble  in  ether, 

*  Palmer  and  Coolidge:  Jour.  Biol.  Chetn.,  17,  251,  1914. 


400  PHYSIOLOGICAL    CHEMISTRY 

acetic  ether,  and  in  water.  Its  solutions  show  characteristic  absorption 
bands  (see  Absorption  Spectra,  Plate  II).  Under  normal  conditions 
urobilin  is  derived  from  the  bile  pigments  in  the  intestine. 

Urobilin  is  increased  in  most  acute  infectious  diseases  such  as  ery- 
sipelas, malaria,  pneumonia,  and  scarlet  fever.  It  is  also  increased  in 
appendicitis,  carcinoma  of  the  liver,  catarrhal  icterus,  pernicious  anemia, 
and  in  cases  of  poisoning  by  antifebrin.  antipyrin,  pyridin,  and  potas- 
sium chlorate.  In  general  it  is  usually  increased  when  blood  destruction 
is  excessive  and  in  disturbances  of  the  liver.  It  is  markedly  decreased 
in  phosphorus  poisoning. 

In  liver  disease,  of  any  type,  urobilinogen  occurs  in  the  urine.  Its 
detection  is  the  basis  of  a  specific  test  ior  functional  liver  incapacity. 

Experiments 

1.  Ammoniacal-zinc  Chloride  Test. — Render  some  of  the  urine  ammoniacal 
by  the  addition  of  ammonixun  hydroxide,  and  after  allowing  it  to  stand  a  short 
time  filter  off  the  precipitate  of  phosphates  and  add  a  few  drops  of  zinc  chloride 
solution  to  the  filtrate.  Observe  the  production  of  a  greenish  fluorescence. 
Examine  the  fluid  by  means  of  the  spectroscope  and  note  the  absorption  band 
which  occupies  much  the  same  position  as  the  absorption  band  of  urobilin  in  acid 
solution  (see  Absorption  Spectra,  Plate  II). 

2.  Ether-absolute  Alcohol  Test. — Mix  urine  and  pure  ether  in  equal  volumes 
and  shake  gently  in  a  separatory  funnel.  Separate  the  ether  extract,  evaporate  it 
to  dryness,  and  dissolve  the  residue  in  2-3  c.c.  of  absolute  alcohol.  Note  the 
greenish  fluorescence.  Examine  the  solution  spectroscopically  and  observe  the 
characteristic  absorption  band  (see  Absorption  Spectra,  Plate  II). 

3.  Ring  Test. — Acidify  25  c.c.  of  urine  with  2-3  drops  of  concentrated  hydro- 
chloric acid,  add  5  c.c.  of  chloroform  and  shake  the  mixture.  Separate  the  chloro- 
form, place  it  in  a  test-tube,  and  add  carefiilly  3-5  c.c.  of  an  alcoholic  solution  of 
zinc  acetate.  Observe  the  formation  of  a  green  ring  at  the  zone  of  contact  of  the 
two  fluids.     If  the  tube  is  shaken  a  fluorescence  may  be  observed. 

4.  Spectroscopic  Examination. — Acidify  the  urine  with  hydrochloric  acid  and 
allow  it  to  remain  exposed  to  the  air  for  a  few  moments.  By  this  means  if  any 
urobilinogen  is  present  it  will  be  transformed  into  urobilin.  The  urine  may  now  be 
examined  by  means  of  the  spectroscope.  If  urobilin  is  present  in  the  fluid  the  char- 
acteristic absorption  band  lying  between  b  and  F  will  be  observed  (see  Absorption 
Spectra,  Plate  II).  It  may  be  found  necessary  to  dUute  the  urine  with  water  before 
a  distinct  absorption  band  is  observed.  This  test  may  be  modified  by  acidifjdng 
10  c.c.  of  urine  with  hydrochloric  acid  and  shaking  it  gently  with  5  c.c.  of  amyl 
alcohol.  The  alcoholic  extract  when  examined  spectroscopically  will  show  the 
characteristic  urobilin  absorption  band.  (Note  the  spectroscopic  examination  in 
experiment  (i)  above.) 

5.  Iodine  Test  (Gerhardt).— To  20  c.c.  of  urine  add  3-5  c.c.  of  chloroform 
and  shake  well.  Separate  the  chloroform  extract  and  add  to  it  a  few  drops  of 
iodine  solution  (I  in  KI).  Render  the  mixture  alkaline  with  dilute  solution  of 
potassium  hydroxide  and  note  the  production  of  a  yellow  or  yellowish-brown  color. 
The  solution  ordinarily  exhibits  a  greenish  fluorescence. 


URIXE  401 

6.  Alcoholic -zinc  Chloride  Test  (Wirsing). — To  20  c.c.  of  urine  add  3-5  c.c. 
of  chloroform  and  shake  gently.  Separate  the  chloroform  extract  and  add  to  it 
a  drop  of  an  alcoholic  solution  of  zinc  chloride.  Note  the  rose-red  color  and  the 
greenish  fluorescence.  If  the  solution  is  turbid  it  may  be  rendered  clear  by  the 
addition  of  a  few  cubic  centimeters  of  absolute  alcohol. 

C.  UROERYTHRIN 

This  pigment  is  frequently  present  in  small  amount  in  normal  urine. 
The  red  color  of  urinary  sediments  is  due  in  great  part  to  the  presence 
of^uroerythrin.  It  is  easily  soluble  in  amyl  alcohol,  slightly  soluble  in 
acetic  ether,  absolute  alcohol,  or  chloroform,  and  nearly  insoluble  in 
water.  Dilute  solutions  of  uroerythrin  are  pink  in  color  while  concen- 
trated solutions  are  orange  red  or  bright  red;  none  of  its  solutions  fluor- 
esce. Uroerythrin  is  increased  in  amount  after  strenuous  physical  exer- 
cise, digestive  disturbances,  fevers,  certain  liver  disorders,  and  in  various 
other  pathological  conditions. 

PTOMAINES  AND  LEUCOMAINES 

These  toxic  substances  are  said  to  be  present  in  small  amount  in 
normal  urine.  Very  little  is  known  definitely,  however,  about  them. 
It  is  claimed  that  five  different  poisons  may  be  detected  in  the  urine, 
and  it  is  further  stated  that  each  of  these  substances  produces  a  spe- 
cific and  definite  symptom  when  injected  intravenously  into  a  rabbit. 
The  resulting  symptoms  are  narcosis,  salivation,  mydriasis,  paralysis, 
and  convulsions.  The  day  urine  is  principally  narcotic  and  is  2-4  times 
as  toxic  as  the  night  urine  which  is  chiefly  productive  of  convulsions. 

PURINE  BASES 

The  purine  bases  found  in  human  urine  are  adenine,  carnine,  epi- 
guanine,  episarkine,  guanine,  xanthine,  heteroxanthine,  hypoxanthine, 
paraxanthine,  and  i-methylxanthine.  The  main  bulk  of  the  purine 
base  content  of  the  urine  is  made  up  of  paraxanthine,  heteroxanthine 
and  i-methylxanthine,  which  are  derived  for  the  most  part  from  the  caf- 
feine, theobromine,  and  theophylline  of  the  food.  The  total  purine 
base  content  is  made  up  of  the  products  of  two  distinct  forms  of  metabo- 
lism, i.e.,  metabolism  of  ingested  nucleins  and  purines  and  metabolism 
of  tissue  nuclein  material.  Purine  bases  resulting  from  the  first  form  of 
metabolism  are  said  to  be  of  exogenous  origin,  whereas  those  resulting 
from  the  second  form  of  metabolism  are  said  to  be  of  endogenous  origin. 
The  daily  output  of  purine  bases  by  the  urine  is  extremely  small  and 
varies  greatly  with  the  individual  (16-60  mg.).  The  output  is  increased 
after  the  ingestion  of  nuclein  material  as  well  as  after  the  increased  de- 
26 


402  PHYSIOLOGICAL   CHEMISTRY 

struction  of  leucocytes.  A  well-marked  increase  accompanies  leukemia. 
Edsall  and  others  have  shown  that  the  output  of  purine  bases  by  the 
urine  is  increased  as  a  result  of  X-ray  treatment.  The  purine  bases 
form  a  higher  percentage  of  the  total  purine  excretion  in  the  case  of 
the  monkey,  sheep  and  goat  than  in  man. 

Experiment 

I.  Formation  of  the  Silver  Salts. — ^Add  an  excess  of  magnesia  mixture ^  to 
25  c.c.  of  urine.  Filter  off  the  precipitate  and  add  ammoniacal  silver  solution^ 
to  the  filtrate.  A  precipitate  composed  of  the  silver  salts  of  the  various  purine 
bases  is  produced.  The  purine  bases  may  be  determined  quantitatively  by  means 
of  Kriiger  and  Schmidt's  method  (see  page  513),  or  Welker's  method  (see  page 
515)- 

2.  Inorganic  Physiological  Constituents 

Ammonia 

Next  to  urea,  ammonia  is  the  most  important  of  the  nitrogenous 
end-products  of  protein  metabolism.  Ordinarily  about  2.5-4.5  per 
cent  of  the  total  nitrogen  of  the  urine  is  eliminated  as  ammonia  and 
on  the  average  this  would  be  about  0.7  gram  per  day.  Under  normal 
conditions  the  ammonia  is  present  in  the  urine  in  the  form  of  the 
chloride,  phosphate,  or  sulphate.  This  is  due  to  the  fact  that  combina- 
tions of  this  sort  are  not  oxidized  in  the  organism  to  form  urea,  but  are 
excreted  as  guch.  This  explains  the  increase  in  the  output  as  ammonia 
which  follows  the  administration  of  the  ammonium  salts  of  the  mineral 
acids  or  of  the  acids  themselves.  On  the  other  hand,  when  ammonium 
acetate  and  many  other  ammonium  salts  of  certain  organic  acids  are 
administered  no  increase  in  the  output  of  ammonia  occurs  since  the  salt 
is  oxidized  and  its  nitrogen  ultimately  appears  in  the  urine  as  urea. 
Acid-forming  foods  (see  page  580)  also  increase  the  ammonia  output, 
whereas  the  administration  of  alkalies  or  of  base-forming  foods  decreases 
the  excretion  of  ammonia 

Experiments^  indicate  that  the  nitrogen  in  food  protein  may  in 
part  be  replaced  by  ammonium  salts. 

Copious  water  drinking  increases  the  ammonia  output.  This  fact 
has  been  interpreted  as  indicating  a  stimulation  of  the  gastric  secretion.^ 

^Magnesia  mixture  may  be  prepared  as  follows:  Dissolve  175  grams  of  MgS04  and 
350  grams  of  NH4CI  in  1400  c.c.  of  distilled  water.  Add  700  grams  of  concentrated  NH4- 
OH,  mix  very  thoroughly  and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

^  Ammoniacal  silver  solution  may  be  prepared  according  to  directions  given  on  page  593. 

^  Grafe  and  Schlapfer:  Zeit.  physio! .  chem.,  77,  i,  191 2,  experiments  by  Abderhalden  in 
same  journal. 

•'Wills  and  Hawk:  Jour.  Am.  Chem.  Soc,  36,  158,  1914. 


TRINE  403 

The  acids  formed  during  the  process  of  protein  destruction  within 
the  body  have  an  influence  upon  the  excretion  of  ammonia  similar  to 
that  exerted  by  acids  which  have  been  administered.  Therefore  a 
pathological  increase  in  the  output  of  ammonia  is  observed  in  such 
diseases  as  are  accompanied  by  an  increased  and  imperfect  protein 
metabolism,  and  especially  in  diabetes,  in  w-hich  disease  acetoacetic 
acid  and  /3-oxybutyric  acid  are  found  in  the  urine  in  combination  with 
the  ammonia. 

Folin  claims  that  a  pronounced  decrease  in  the  extent  of  protein 
metabolism,  as  measured  by  the  total  nitrogen  in  the  urine,  is  frequently 
accompanied  by  a  decreased  elimination  of  ammonia.  The  ammonia 
elimination  is  therefore  probably  determined  by  other  factors  than  the 
total  protein  catabolism  as  such.  Furthermore,  he  believes  that  a 
decided  decrease  in  the  total  nitrogen  excretion  is  always  accompanied 
by  a  relative  increase  in  the  ammonia-nitrogen,  provided  the  food  is  of  a 
character  yielding  an  alkaline  ash. 

The  quantitative  determination  of  ammonia  must  be  made  upon 
the  fresh  urine,  since  upon  standing  the  normal  urine  will  undergo  am- 
moniacal  fermentation  (see  page  362). 

Experiments 
(See  Experiment  2  under  Phosphates,  page  408.) 

Sulphates 

.  Sulphur  in  combination  is  excreted  in  two  forms  in  the  urine:  first, 
as  unoxidized,  loosely  combined  or  neutral  sulphur,  and  second,  as  oxidized 
or  acid  sulphur.  The  unoxidized  or  neutral  sulphur  is  excreted  mainly 
as  a  constituent  of  such  bodies  as  cystine,  cysteine,  taurine,  hydrogen 
sulphide,  ethyl  sulphide,  thiocyanates,  sulphonic  acids,  oxyproteic  acid, 
alloxyproteic  acid,  and  uroferric  acid.  The  amount  of  neutral  sulphur 
eliminated  is  in  great  measure  independent  of  the  extent  of  protein 
decomposition  or  of  the  total  sulphur  excretion.  In  this  characteristic 
it  is  somewhat  similar  to  the  excretion  of  creatinine.  The  oxidized 
sulphur  is  eliminated  in  the  form  of  sulphuric  acid,  principally  as  salts  of 
sodium,  potassium,  calcium,  and  magnesium;  a  relatively  small  amount 
occurs  in  the  form  of  ethereal  sulphuric  acid,  i.e.,  sulphuric  acid  in  com- 
bination with  such  orofnatic  bodies  as  phenol,  indole,  skatole.  cresol, 
pyrocatechol,  and  hydroquinol.  Sulphuric  "acid  in  combination  with 
Na,  K,  Ca  or  Mg  is  sometimes  termed  inorganic  or  preformed  sulphuric 
acid,  whereas  the  ethereal  sulphuric  acid  is  sometimes  called  conjugate 
sulphuric  acid.     The  greater  part  of  the  sulphur  is  eliminated  in  the 


404  PHYSIOLOGICAL  CHEMISTRY 

oxidized  form,  but  the  absolute  percentage  of  sulphur  excreted  as  the 
performed,  ethereal  or  loosely  combined  type  depends  upon  the  total 
quantity  of  sulphur  present;  i.e.,  there  is  no  definite  ratio  between 
the  three  forms  of  sulphur  which  will  apply  under  all  conditions.  The 
preformed  sulphuric  acid  may  be  precipitated  directly  from  acidified 
urine  with  BaCl2,  whereas  the  ethereal  sulphuric  acid  must  undergo  a 
preliminary  boiling  in  the  presence  of  a  mineral  acid  before  it  can  be 
so  precipitated. 

The  sulphuric  acid  excreted  in  the  urine  arises  principally  from 
the  oxidation  of  protein  material  within  the  body;  a  relatively  small 
amount  is  due  to  ingested  sulphates.  Under  normal  conditions  about 
2.5  grams  of  sulphuric  acid  (SO3)  are  eliminated  daily,  about  75-95  per 
cent  of  this  being  in  the  form  of  sulphates.  About  90  per  cent  of  this 
sulphate  excretion  is  in  the  form  of  inorganic  sulphate  and  10  per  cent  as 
ethereal  sulphates.  Since  the  sulphuric  acid  content  of  the  urine  has, 
for  the  most  part,  a  protein  origin  and  since  one  of  the  most  important 
constituents  of  the  protein  molecule  is  nitrogen,  it  would  be  reasonable 
to  suppose  that  a  fairly  definite  ratio  might  exist  between  the  excretion 
of  these  two  elements.  However,  when  we  appreciate  that  the  per- 
centage content  of  N  and  S  present  in  different  proteins  is  subject  to 
rather  wide  variations,  the  fixing  of  a  ratio  which  will  express  the  exact 
relation  existing  between  these  two  elements  as  they  appear  in  the  urine 
as  end-products  of  protein  metabolism  is  practically  impossible.  It 
has  been  suggested  that  the  ratio  5 :  i  expresses  this  relation  in  a  general 
way. 

Pathologically,  the  excretion  of  sulphuric  acid  by  the  urine  is  in- 
creased in  acute  fevers  and  in  all  other  diseases  marked  by  a  stimulated 
metabolism,  whereas  a  decrease  in  the  sulphuric  acid  excretion  is  observed 
in  those  diseases  which  are  accompanied  by  a  loss  of  appetite  and  a 
diminished  metabolic  activity. 

Experiments 

1.  Detection  of  Inorganic  Sulphuric  Acid. — Place  about  10  c.c.  of  urine  in  a 
test-tube,  acidify  with  acetic  acid  and  add  some  barium  chloride  solution.  A 
white  precipitate  of  barium  sulphate  forms. 

2.  Detection  of  Ethereal  Sulphuric  Acid. — Filter  off  the  barium  sulphate 
precipitate  formed  in  the  above  experiment,  add  i  c.c.  of  hydrochloric  acid  and  a 
little  barium  chloride  solution  to  the  filtrate  and  heat  the  mixture  to  boiling  for 
1-2  minutes.  Note  the  appearance  of  a  turbidity  due  to  the  presence  of  sul- 
phuric acid  which  has  been  separated  from  the  ethereal  sulphates  and  has  com- 
bined with  the  barium  of  the  BaClo  to  form  BaS04. 

3.  Detection  of  Unoxidized  or  Neutral  Sulphur. — Place  about  10  c.c.  of  urine 
in  a  test-tube,  introduce  a  small  piece  of  zinc,  add  sufficient  hydrochloric  acid 


URINE 


405 


Fig.  1 28. — Calcium  Sulphate. 
(Hensel  and  Weil.) 


to  cause  a  gentle  evolution  of  hydrogen,  and  over  the  mouth  of  the  tube  place  a 
filter  paper  saturated  with  lead  acetate  solution.  In  a  short  time  the  portion  of 
the  paper  in  contact  with  the  vapors  within  the  test-tube  becomes  blackened  due 
to  the  formation  of  lead  sulphide.  The  nascent  hydrogen  has  reacted  with  the 
loosely  combined  or  neutral  stilphur  to  form  hydrogen  sulphide,  and  this  gas  com- 
ing in  contact  with  the  lead  acetate  paper  has  caused  the  production  of  the  black 
lead  sulphide.  Sulphur  in  the  form  of  inorganic  or  ethereal  sulphuric  acid  does 
not  respond  to  this  test. 

4.  Calciimi  Sulphate  Crystals. — Place  10  c.c.  of  urine  in  a  test-tube,  add  10 
drops  of  calcium  chloride  solution  and  allow  the  tube  to  stand  until  crystals  form. 
Examine  the  calcium  sulphate  crystals  under 

the  microscope  and  compare  them  with  those  ^^  'JJj^'       *    A  "V 

shown  in  Fig.  128.  .    '.'^         . . 

Chlorides 

Next  to  urea,  the  chlorides  consti- 
tute the  chief  solid  constituent  of  the 
urine.  The  principal  chlorides  found 
in  the  urine  are  those  of  sodium,  potas- 
sium, ammonium,  and  magnesium,  with 
sodium   chloride    predominating.     The  r,\ 

excretion  of  chloride  is  dependent,  in  great  part,  upon  the  nature  of  the 
diet,  but  on  the  average  the  daily  output  is  about  10-15  grams,  expressed 
as  sodium  chloride.  Copious  water  drinking  increases  the  output  of 
chlorides  considerably.  Because  of  their  solubility,  chlorides  are  never 
found  in  the  urinary  sediment. 

Since  the  amount  of  chlorides  excreted  in  the  urine  is  due  primarily 
to  the  chloride  content  of  the  food  ingested,  it  follows  that  a  decrease 
in  the  amount  of  ingested  chloride  will  likewise  cause  a  decrease  in  the 
chloride  content  of  the  urine.  In  cases  of  actual  fasting  the  chloride 
content  of  the  urine  may  be  decreased  to  a  slight  trace  which  is  derived 
from  the  body  fluids  and  tissues.  Under  these  conditions,  however, 
an  examination  of  the  blood  of  the  fasting  subject  will  show  the  per- 
centage of  chlorides  in  this  fluid  to  be  approximately  normal.  This 
forms  a  very  striking  example  of  the  care  nature  takes  to  maintain  the 
normal  composition  of  the  blood.  There  is  a  limit  to  the  power  of  the 
body  to  maintain  this  equilibrium,  however,  and  if  the  fasting  organism 
be  subjected  to  the  influence  of  diuretics  for  a  time,  a  point  is  reached 
where  the  normal  composition  of  the  blood  can  no  longer  be  maintained 
and  a  gradual  decrease  in  its  chloride  content- occurs  which  tinally  results 
in  death.  Death  is  supposed  to  result  not  so  much  because  of  a  lack  of 
chlorine  as  from  a  deficiency  of  sodium.  This  is  shown  from  the  fact  that 
potassium  chloride,  for  instance,  cannot  replace  the  sodium  chloride 


4o6  PHYSIOLOGICAL   CHEMISTRY 

of  the  blood  when  the  latter  is  decreased  in  the  manner  above  stated. 
When  this  substitution  is  attempted  the  potassium  salt  is  excreted  at 
once  in  the  urine,  and  death  follows  as  above  indicated. 

Pathologically  the  excretion  of  chlorides  may  be  decreased  in  some 
fevers,  chronic  nephritis,  croupous  pneumonia,  diarrhoea,  certain  stom- 
ach disorders,  and  in  acute  articular  rheumatism.  Any  condition  ac- 
companied by  the  formation  of  an  exudate  {e.g.,  pneumonia)  will  cause 
a  diminished  chloride  output.  In  convalescence  and  with  resolution 
of  the  exudate  the  chloride  excretion  rises  again. 

Experiment 

Detection  of  Chlorides  in  Urine. — Place  about  5  c.c.  of  xxrine  in  a  test-tube, 
render  it  acid  with  nitric  acid  and  add  a  few  drops  of  a  solution  of  silver  nitrate. 
A  white  precipitate,  due  to  the  formation  of  silver  chloride,  is  produced.  This 
precipitate  is  soluble  in  ammonium  hydroxide. 

Phosphates 

Phosphoric  acid  exists  in  the  urine  in  two  general  forms:  First, 
that  in  combination  with  the  alkali  metals,  sodium  and  potassium, 
and  the  radical  ammonium;  second,  that  in  combination  with  the 
alkaline  earth  metals,  calcium  and  magnesium.  Phosphates  formed 
through  a  union  of  phosphoric  acid  with  the  alkali  metals  are  termed 
alkaline  phosphates,  or  phosphates  of  the  alkali  metals,  whereas  phos- 
phates formed  through  a  union  of  phosphoric  acid  with  the  alkaline 
earth  metals  are  termed  earthy  phosphates,  or  phosphates  of  the  alkaline 
earth  metals. 

Three  series  of  salts  are  formed  by  phosphoric  acid:  Normal, 
M3PO4/  mono-hydrogen,  M2HPO4,  and  di-hydrogen,  MH2PO4.  The  di- 
hydrogen  salts  are  acid  in  reaction,  and  it  is  claimed  that  about  60  per 
cent  of  the  total  phosphate  content  of  the  urine  is  in  the  form  of  this 
type  of  salt,  and  that  the  acidity  of  the  urine  is  due  in  great  part  to  the 
presence  of  sodium  di-hydrogen  phosphate  (see  page  361).  Henderson- 
maintains  that  "determinations  of  hydrogen  ionization  in  urine  and  its 
behavior  toward  indicators  both  support  the  view  that  in  urine  there 
exists  a  mixture  of  mono-  and  di-hydrogen  phosphates  of  sodium, 
ammonium  and  other  bases." 

In  bones  the  phosphates  occur  principally  in  the  form  of  the  normal 
salts  of  calcium  and  magnesium.  The  mono-hydrogen  salts  as  a  class 
are  alkaline  in  reaction  to  litmus,  and  it  is  to  the  presence  of  di-sodium 

1  M  may  be  occupied  by  any  of  the  alkali  metals  or  alkaline  earth  metals. 
*  Henderson:  Am.  Jour.  Physiol.,  15,  257,  1906. 


URINE  407 

hydrogen  phosphate,  Xa2HP04,  that  the  greater  part  of  the  alkalinity 
of  the  saliva  is  due. 

The  excretion  of  phosphoric  acid  is  extremely  variable,  but  on  the 
average  the  total  output  for  24  hours  is  about  2.5  grams,  expressed 
asP205.  Ordinarily  the  total  output  is  mainly  in  the  form  of  phosphates 
and  is  distributed  between  alkaline  phosphates  and  earthy  phosphates 
approximately  in  the  ratio  2:1.  The  organic  phosphorus  of  the  urine 
constitutes  only  1-4  per  cent  of  the  total  phosphorus  content.  The 
greater  part  of  this  phosphoric  acid  arises  from  the  ingested  food,  either 
from  the  preformed  phosphates  or  more  especially  from  the  phosphorus 
in  organic  combination  such  as  we  find  it  in  phospho-proteins,  nucleo- 
proteins  and  lecithins;  the  phosphorus-containing  tissues  of  the  body 
also  contribute  to  the  total  output  of  this  element.  Alkaline  phosphates 
ingested  with  the  food  have  a  tendency  to  increase  the  phosphoric  acid 
content  of  the  urine  to  a  greater  extent  than  the  earthy  phosphates  so 
ingested.  This  is  due,  in  a  measure,  to  the  fact  that  a  portion  of  the 
earthy  phosphates,  under  certain  conditions,  may  be  precipitated  in  the 
intestine  and  excreted  in  the  feces;  this  is  especially  to  be  noted  in  the 
case  of  herbivorous  animals.  Since  the  extent  to  which  the  phosphates 
are  absorbed  in  the  intestine  depends  upon  the  form  in  which  they  are 
present  in  the  food,  under  ordinary  conditions,  there  can  be  no  absolute 
relationship  between  the  urinary  output  of  nitrogen  and  phosphorus. 
If  the  diet  is  constant,  however,  from  day  to  day,  thus  allowing  of  the 
preparation  of  both  a  nitrogen  and  a  phosphorus  balance,^  a  definite 
ratio  may  be  established.  In  experiments  upon  dogs  which  were  fed 
an  exclusive  meat  diet,  the  ratio  of  nitrogen  to  phosphorus,  in  the  urine 
and  feces,  was  found  to  be  8.1  :  i. 

It  has  been  demonstrated  by  recent  investigation  that  the  ingestion  of 
inorganic  phosphorus  compounds  may  give  rise  to  organic  phosphorus 
compounds  such  as  lecithin,  phosphatides,  nucleoproteins  and  phospho- 
proteins.  This  is  an  instance  of  an  organic  substance  synthesized  from 
an  inorganic  substance.  The  experiments  have  been  made  principally 
on  ducks-  and  hens.'' 

Pathologically  the  excretion  of  phosphoric  acid  is  increased  in  such 
diseases  of  the  bones  as  diffuse  periostosis,  osteomalacia,  and  rickets ; 
according  to  some  investigators,  in  the  early  stages  of  pulmonary  tuber- 
culosis, in  acute  yellow  atrophy  of  the  liver,  in  diseases  which  are  ac- 
companied by  an  extensive  decomposition  of  nervous  tissue,  and  after 

^  In  metabolism  experiments,  a  statement  showing  the  relation  existing  between  the 
nitrogen  content  of  the  food  on  the  one  hand  and  that  of  the  urine  and  feces  on  the  other, 
for  a  definite  period,  is  termed  a  nitrogen  balance  or  a  "balance  of  the  income  and  outgo  of 
nitrogen"  (see  chapter  on  Metabolism). 

*Fingerling:  Biochem.  Zeit.,  38,  448,  191 2. 

'McCollum  and  Halpin:  Jour.  Biol.  Chem.,  11,  47  (Proceedings),  1912. 


4o8 


PHYSIOLOGICAL    CHEMISTRY 


sleep  induced  by  potassium  bromide  or  chloral  hydrate  (Mendel).  It 
is  also  increased  after  copious  water  drinking.  A  decrease  in  the 
excretion  of  phosphates  is  at  times  noted  in  febrile  affections,  such  as 
the  acute  infectious  diseases,  in  pregnancy,  in  the  period  during  which 
the  fetal  bones  are  forming,  and  in  diseases  of  the  kidneys,  because  of 
non-elimination. 

The  so-called  "phosphaturias"  many  times  represent  decreased 
acidity  and  not  increased  phosphate  content  of  the  urine.  Such  con- 
ditions are,  however,  significant  as  indicating  a  possible  tendency  to  the 
formation  of  phosphatic  calculi. 

Experiments 

I.  Formation  of  "Triple  Phosphate." — Place  some  urine  in  a  beaker,  render 
it  alkaline  with  ammonium  hydroxide,  add  a  small  amount  of  magnesimn  sul- 


FiG.  129. — "Triple  Phosphate."     (Ogden.) 


phate  solution  and  allow  the  beaker  to  stand  in  a  cool  place  over  night.  Crystals 
of  ammonium  magnesiiun  phosphate,  "triple  phosphate,"  form  under  these  con- 
ditions. Examine  the  crystalline  sediment  under  the  microscope  and  compare 
the  forms  of  the  crystals  with  those  shown  in  Fig.  129,  above. 

2.  Ammoniacal  Fermentation. — Stand  some  urine  aside  in  a  beaker  for 
several  days.  Ammoniacal  fermentation  will  develop  and  "triple  phosphate" 
crystals  will  form. 

(a)  Examine  the  sediment  under  the  microscope  and  compare  the  crystals  with 
those  shown  in  Fig.  129. 

(b)  Hold  a  glass  rod  dipped  in  concentrated  hydrochloric  acid  near  the 
surface  of  the  urine.     Note  the  fumes  of  ammoniiun  chloride. 

(c)  Insert  a  strip  of  red  litmus  paper  in  the  urine.  Permit  the  paper  to  dry. 
Note  the  gradual  restoration  of  red  color,  due  to  volatiUzation  of  ammonia 
(volatile  alkaU).    Run  a  control  test  using  0.5  per  cent  Na2C03  (fixed  alkali). 

3.  Detection  of  Earthy  Phosphates. — Place  10  c.c.  of  urine  in  a  test-tube  and 
render  it  alkaline  with  ammonium  hydroxide.  Warm  the  mixture  and  note  the 
separation  of  a  precipitate  of  earthy  phosphates. 


URINE  409 

4.  Detection  of  Alkaline  Phosphates. — Filter  off  the  earthy  phosphates  as 
formed  in  the  last  experiment,  and  add  a  small  amount  of  magnesia  mixture  'see 
page  602)  to  the  filtrate.  Now  warm  the  mixture  and  observe  the  formation  of  a 
white  precipitate  due  to  the  presence  of  alkaline  phosphates.  Note  the  difference 
in  the  size  of  the  precipitates  of  the  two  forms  of  phosphates  from  this  same  vol- 
vune  of  urine.  Which  form  of  phosphates  was  present  in  the  larger  amount, 
earthy  or  alkaline? 

5.  Influence  upon  Fehhng's  Solution. — Place  2  c.c.  of  Fehhng's  solution  in  a 
test-tube,  dilute  it  with  4  volumes  of  water  and  heat  to  boiling.  Add  a  solution 
of  sodium  dihydrogen  phosphate,  NaHjP04,  a  small  amount  at  a  time,  and  heat 
after  each  addition.  What  do  you  observe?  What  does  this  observation  force 
you  to  conclude  regarding  the  interference  of  phosphates  in  the  testing  of  dia- 
betic urine  by  means  of  Fehhng's  test? 

Sodium  and  Potassium 

The  elements  sodium  and  potassium  are  always  present  in  the  urine. 
Usually  they  are  combined  with  such  acidic  radicals  as  CI,  CO3,  SO4 
and  PO4.  The  amount  of  potassium,  expressed  as  KoO,  excreted  in 
24  hours  by  an  adult,  subsisting  upon  a  mixed  diet,  is  on  the  average 
2-3  grams,  whereas  the  amount  of  sodium,  expressed  as  Na20,  under 
the  same  conditions,  is  ordinarily  4-6  grams.  The  ratio  of  K  to  Na  is 
generally  about  3  : 5.  The  absolute  quantity  of  these  elements  excreted 
depends,  of  course,  in  large  measure  upon  the  nature  of  the  diet.  Be- 
cause of  the  non-ingestion  of  XaCl  and  the  accompanying  destruction 
of  potassium-containing  body  tissues,  the  urine  during  fasting  contains 
more  potassium  salts  than  sodium  salts. 

Pathologically  the  output  of  potassium,  in  its  relation  to  sodium, 
may  be  increased  during  fever;  following  the  crisis,  however,  the  out- 
put of  this  element  may  be  decreased.  It  may  also  be  increased  in 
conditions  associated  with  acidosis. 

Calcium  and  Magnesium 

The  greater  part  of  the  calcium  and  magnesium  excreted  in  the 
urine  is  in  the  form  of  phosphates.  The  daily  output  of  calcium,  which 
depends  principally  upon  the  nature  of  the  diet,  aggregates  on  the 
average  about  0.1-0.4  gram  (expressed  as  CaO)  per  day.  The  per- 
centage of  calcium  salts  present  in  the  urine  at  any  one  time  (10-40 
per  cent  of  total  calcium  output)  forms  no  dependable  index  as  to  the 
absorption  of  this  class  of  salts,  since  they  are  again  excreted  into  the 
intestine  after  absorption.  It  is  therefore  impossible  to  draw  any  satis- 
factory conclusions  regarding  the  excretion  of  calcium  unless  we  obtain 
accurate  analytical  data  from  both  the  feces  and  the  urine. 


4IO  PHYSIOLOGICAL   CHEMISTRY 

Very  little  is  known  positively  regarding  the  actual  course  of  the 
excretion  of  the  calcium  under  pathological  conditions.  An  excess 
is  found  in  some  diseases  of  the  bones,  e.g.,  osteomalacia.  In  others 
as  in  rickets  the  urinary  excretion  may  be  very  low. 

The  daily  excretion  of  magnesium  by  way  of  the  urine  usually 
amounts  to  between  o.i  and  0.3  gram,  expressed  as  MgO.  The  amount 
depends  mainly  on  the  diet.  About  50  per  cent  or  more  of  the  excreted 
magnesium  is  usually  eliminated  by  the  kidneys,  the  remainder  passes 
out  in  the  feces.  There  may  be  a  retention  of  magnesium  in  certain 
bone  disorders  accompanying  a  loss  of  calcium;  in  osteomalacia  for 
example.  Thus  the  excretion  of  calcium  and  magnesium  do  not  neces- 
sarily run  parallel. 

Carbonates 

Carbonates  generally  occur  in  small  amount  in  the  urine  of  man 
and  carnivora  under  normal  conditions,  whereas  much  larger  quanti- 
ties are  ordinarily  present  in  the  urine  of  herbivora.  The  alkaline 
reaction  of  the  urine  of  herbivora  is  dependable  in  great  measure  upon 
the  presence  of  carbonates.  In  general  a  urine  containing  carbonates 
in  appreciable  amount  is  turbid  when  passed  or  becomes  so  shortly 
after.  These  bodies  ordinarily  occur  as  alkali  or  alkaline  earth  com- 
pounds and  the  turbid  character  of  urine  containing  them  is  usually 
due  principally  to  the  latter  class  of  substances.  The  carbonates  of 
the  alkaline  earths  are  often  found  in  amorphous  urinary  sediments. 

Iron 

Iron  is  present  in  small  amount  in  normal  urine.  It  probably  occurs 
partly  in  inorganic  and  partly  in  organic  combination.  The  iron  con- 
tained in  urinary  pigments  or  chromogens  is  in  organic  combination. 
According  to  different  investigators  the  iron  content  of  normal  urine  will 
probably  not  average  more  than  1-5  mg.  per  day.  After  splenectomy 
there  is  an  increased  loss  of  iron  from  the  body  particularly  by  way  of 
the  feces  (Asher). 

Experiment 

Detection  of  Iron  in  Urine. — Evaporate  a  convenient  volume  (10-15  c.c.)  of 
urine  to  dryness.  Incinerate  and  dissolve  the  residue  in  a  few  drops  of  iron-free 
hydrochloric  acid  and  dilute  the  acid  solution  with  5  c.c.  of  water.  Divide  the 
acid  solution  into  two  parts  and  make  the  following  tests:  (a)  To  the  first  part  add 
a  solution  of  ammonium  thiocyanate;  a  red  color  indicates  the  presence  of  iron. 
(b)  To  the  second  part  of  the  solution  add  a  little  potassium  ferrocyanide  solution; 
a  precipitate  of  Prussian  blue  forms  upon  standing. 


URJNE  411 

Fluorides,  Nitrates,  Silicates  and  Hydrogen  Peroxide 

These  substances  are  all  found  in  traces  in  human  urine  under  nor- 
mal conditions.  Nitrates  are  undoubtedly  introduced  into  the  organ- 
ism in  the  water  and  ingested  food.  The  average  excretion  of  nitrates 
is  about  0.5  gram  per  day,  the  output  being  the  largest  upon  a  vegeta- 
ble diet  and  smallest  upon  a  meat  diet.  Nitrites  are  found  only  in 
urine  which  is  undergoing  decomposition  and  are  formed  from  nitrates 
in  the  course  of  ammoniacal  fermentation.  Hydrogen  peroxide  has 
been  detected  in  the  urine,  but  its  presence  is  believed  to  possess  no 
pathological  importance. 


CHAPTER  XXIII 
URINE:  PATHOLOGICAL  CONSTITUENTS^ 


Glucose. 


Proteins 


Blood 

Pus. 

Bile. 


Serum  albumin. 
Serum  globulin. 

Proteoses. 


Peptone. 

Nucleoprotein. 

Fibrin. 
I  Oxyhemoglobin. 
Form  elements. 
Pigment. 


Deutero-proteose. 
Hetero-proteose. 
"Bence-Jones'  protein.' 


J  Pigments. 

I  Acids. 
Creatine.^ 
Acetone. 
Acetoacetic  acid. 
/3-Hydroxybutyric  acid. 
Conjugate  glycuronates. 
Pentoses. 
Fat. 

Hematoporphyrin. 
Lactose. 
Galactose. 
Fructose. 
Arsenic. 
Mercury. 
Inositol. 
Laiose. 
Melanin. 
Urorosein. 
Nephrorosein. 
Urochromogen. 
Unknown  substances. 

1  See  note  at  the  bottom  of  page  369. 
"  Normal  constituent  of  urine  of  infants  and  children. 

412 


URINE  413 

GLUCOSE 

Traces  of  this  sugar  occur  in  normal  urine,  but  the  amount  is  not 
sufficient  to  be  readily  detected  by  the  ordinary  simple  qualitative 
tests.  There  are  two  distinct  types  of  pathological  glycosuria,  i.e, 
transitory  glycosuria  and  persistent  glycosuria.  The  transitory  type 
may  follow  the  ingestion  of  an  excess  of  sugar,  causing  the  assimilation 
limit  to  be  exceeded,  or  it  may  accompany  any  one  of  several  disorders 
which  cause  impairment  of  the  power  of  assimilating  sugar.  In  the 
persistent  type  large  amounts  of  sugar  are  excreted  daily  in  the  urine 
for  long  periods  of  time.  Under  such  circumstances  a  condition  known 
as  diabetes  mellitus  exists.  In  this  disorder  the  urine  may  contain  10 
per  cent  of  glucose  and  the  average  sugar  content  is  3-5  per  cent. 
Ordinarily,  diabetic  urine  which  contains  a  high  percentage  of  sugar 
possesses  a  faint  yellow  color,  a  high  specific  gravity,  and  a  volume 
which  is  above  normal.  Over  100  grams  of  sugar  are  daily  eliminated 
in  some  severe  cases  of  diabetes  mellitus. 

Experiments 

The  various  tests  for  glucose  in  the  urine  which  are  embraced  in  the 
experiments  given  herewith  are  based  upon  one  of  the  following 
properties  of  this  sugar: 

(i)  Its  power  to  reduce  the  oxides  of  certain  metals  in  alkaline  solution. 

(2)  Its  power  to  rotate  the  plane  of  polarized  light. 

(3)  Its  power  to  form  crystalline  osazones  with  phenylhydrazine. 

(4)  Its  ability  to  ferment  with  ordinary  yeast. 

I.  Phenylhydrazine  Reaction. — Test  the  urine  according  to  one  of  the  fol- 
lowing methods :  (a)  To  a  small  amount  of  phenylhydrazine  mixture  (enough  to 
fill  the  rounded  portion  of  a  small  test-tube),  furnished  by  the  instructor,^  add 
5  c.c.  of  the  urine,  shake  well,  and  heat  on  a  boiling  water-bath  for  one-half  to 
three-quarters  of  an  hour.  Allow  the  tube  to  cool  slowly  (not  imder  the  tap)  and 
examine  the  crystals  microscopically  (Plate  III,  opposite  page  22).  If  the  solu- 
tion has  become  too  concentrated  in  the  boiling  process  it  will  be  Ught  red  in  color 
and  no  crystals  will  separate  until  it  is  diluted  with  water. 

Yellow  crystalline  bodies  called  osazones  are  formed  from  certain 
sugars  under  these  conditions,  in  general  each  individual  sugar  giving 
rise  to  an  osazone  of  a  definite  crystalline  form  which  is  typical  for  that 
sugar. 

It  is  important  to  remember  in  this  connection  that,  of  the  simple 
sugars  of  interest  in  physiological  chemistry,  glucose  and  fructose  yield 
the  same  osazone,  with  phenylhydrazine.     Eachozazone  has  a  definite 

1  This  mixture  is  prepared  by  combining  one  part  of  phenylhydrazinc-hydrochloride  and 
two  parts  of  sodium  acetate,  by  uriglit.     These  are  thoroughly  mi.xed  in  a  mortar. 


414 


PHYSIOLOGICAL   CHEMISTRY 


melting-point,  and  as  a  further  and  more  accurate  means  of  identifica- 
tion it  may  be  recrystallized  and  identified  by  the  determination  of  its 
melting-point  and  nitrogen  content.  The  reaction  taking  place  in  the 
formation  of  phenylglucosazone  is  as  follows: 

CH2OH  CH2OH 


(CH0H)3 
CHOH 


+C6HbNH-NH2- 


Glue 
CH2OH 


Glucose 


Phenylhydrazine 


(CH0H)3 

+C6H5NH-NH2- 
CHOH 
yN-NHCeHe+HaO 


Phenylhydrazone 

CH2OH 


(CH0H)3 

!  +C6H5NH-NH2-^ 

C  =  0 

|^N-NHC6H5+C6H5NH2+NH3 

C 

Aniline 


(CH0H)3 

C  =  N-NHC6H5+H20 
I^N-NHCeHs 

C 

Glucosazone 


(b)  Place  5  c.c.  of  the  urine  in  a  test-tube,  add  i  c.c.  of  phenylhydrazine- 
acetate  solution  fiunished  by  the  instructor/  and  heat  on  a  boiling  water-bath 
for  one-half  to  three-quarters  of  an  hour.  Allow  the  Uquid  to  cool  slowly  and 
examine  the  crystals  microscopically  (Plate  III,  opposite  page  22). 

The  phenylhydrazine  test  has  been  so  modified  by  Cipollina  as  to 
be  of  use  as  a  rapid  clinical  test.  The  directions  for  this  test  are  given 
in  the  next  experiment. 

2.  CipoUina's  Test. — Thoroughly  mix  4  c.c.  of  urine,  5  drops  of  phenylhydrazine 
(the  base)  and  yi.  c.c.  of  glacial  acetic  acid  in  a  test-tube.  Heat  the  mixture  for 
about  one  minute  over  a  low  flame,  shaking  the  tube  continually  to  prevent  loss  of 
fluid  by  bumping.  Add  4-5  drops  of  potassium  hydroxide  or  sodium  hydroxide 
(sp.  gr.  1. 1 6),  being  certain  that  the  fluid  in  the  test-tube  remains  acid;  heat  the 
mixture  again  for  a  moment  and  then  cool  the  contents  of  the  tube.  Ordinarily 
the  crystals  form  at  once,  especially  if  the  urine  possesses  a  low  specific  gravity. 
If  they  do  not  appear  immediately  allow  the  tube  to  stand  at  least  20  minutes  before 
deciding  upon  the  absence  of  sugar. 

Examine  the  crystals  under  the  microscope  and  compare  them  with  those  shown 
in  Plate  III,  opposite  page  22. 

3.  Riegler's  Reaction.'* — Introduce  o.i  gram  of  phenylhydrazine-hydrochloride 
and  0.25  gram  of  sodium  acetate  into  a  test-tube,  add  20  drops  of  the  urine  under 

^  This  solution  is  prepared  by  mixing  one  part  by  volume,  in  each  case  of  glacial  acetic 
acid,  one  part  of  water  and  two  parts  of  phenylhydrazine  (the  base). 
^Riegler:  Compt.  rend.  soc.  biol.,  66,  p.  795. 


URINE  415 

examination,  and  heat  the  mixture  to  boiling.  Now  introduce  10  c.c.  of  a  3  per 
cent  solution  of  potassium  hydroxide  and  gently  shake  the  tube  and  contents.  If 
the  urine  under  examination  contains  glucose  the  liquid  in  the  tube  will  assume  a 
red  color.  One  per  cent  glucose  yields  an  immediate  color  whereas  0.05  per  cent 
yields  the  color  only  after  the  lapse  of  a  period  of  one-half  hour  from  the  time  the 
alkali  is  added.  If  the  color  appears  after  the  30-minute  interval  the  color  change 
is  without  significance  inasmuch  as  sugar-free  urines  will  respond  thus.  The  reac- 
tion is  given  by  all  aldehydes  and  therefore  the  test  cannot  be  safely  employed  in 
testing  urines  preserved  by  formaldehyde.  Albumin  does  not  interfere  with  the 
test. 

4.  Bottu's  Test.^ — To  8  c.c.  of  Bottu's  reagent-  in  a  test-tube  add  i  c.c.  of  the 
urine  under  examination  and  mix  the  liquids  by  gentle  shaking.  Now  heat  the 
upper  portion  of  the  mixture  to  boiling,  add  an  additional  i  c.c.  of  urine  and  heat 
the  mixture  again  immediately.  The  appearance  of  a  blue  color  accompanied  by 
the  precipitation  of  small  particles  of  indigo  blue  indicates  the  presence  of  glucose 
in  the  urine  under  examination.  The  test  will  serve  to  detect  the  presence  of  o.i 
per  cent  of  glucose  and  is  uninfluenced  by  creatinine  or  by  ammonium  salts. 

5.  Reduction  Tests. — To  their  aldehyde  or  ketone  structure  many 
sugars  owe  the  property  of  readily  reducing  the  alkaline  solutions  of  the 
oxides  of  metals  like  copper,  bismuth,  and  mercury;  they  also  possess 
the  property  of  reducing  ammoniacal  silver  solutions  vi^ith  the  separa- 
tion of  metallic  silver.  Upon  this  property  of  reduction  the  most  widely 
used  tests  for  sugars  are  based.  When  whitish-blue  cupric  hydroxide  in 
suspension  in  an  alkaline  liquid  is  heated  it  is  converted  into  insoluble 
black  cupric  oxide,  but  if  a  reducing  agent  like  certain  sugars  be  present 
the  cupric  hydroxide  is  reduced  to  insoluble  yellow  cuprous  hydroxide, 
which  in  turn  on  further  heating  may  be  converted  into  brownish-red  or 
red  cuprous  oxide.     These  changes  are  indicated  as  follows: 

OH 

/ 
Cu        -^Cu  =  0+H20. 

\  Cupric  oxide 

\  (black). 

OH 

Cupric  hydroxide 
(whitish-blue). 

OH 

/ 
Cu 

\ 
OH 

-^  2Cu-0H-f  HoO+0. 


/^XJ       Cuprous  hydroxide 
^•"-  (yellow). 


Cu 

\ 
OH 

^  Bottu:  Cpmpl.  rend.  soc.  bioL,  66,  p.  972. 

*This  reagent  contains  3.5  grams  of  c-nitrophenylpropiolic  acid  and  5  c.c.  of  a  freshly 
prepared  10  per  cent  solution  of  sodium  hydro.xide  per  liter. 


41 6  PHYSIOLOGICAL    CHEMISTRY 

Cu 


Cu— OH 
Cu— OH 


0+H20. 


Cu    ■ 

Cuprous  hydroxide        Cuprous  oxide 
(yellow).  (brownish-red). 

The  chemical  equations  here  discussed  are  exempHfied  in  Trommer's 
and  Fehling's  tests. 

(a)  Trommer^s  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  one-half  its  volume 
of  KOH  or  NaOH.  Mix  thoroughly  and  add,  drop  by  drop,  agitating  after  the 
addition  of  each  drop,  a  very  dilute  solution  of  copper  sidphate.  Continue  the  addi- 
tion until  there  is  a  slight  permanent  precipitate  of  cupric  hydroxide  and  in  conse- 
quence the  solution  is  slightly  turbid.  Heat,  and  the  cupric  hydroxide  is  reduced 
to  yellow  cuprous  hydroxide  or  to  brownish-red  cuprous  oxide. 

If  the  solution  of  copper  sulphate  used  is  too  strong,  a  small  brownish-red  pre- 
cipitate produced  in  the  presence  of  a  low  percentage  of  glucose  may  be  entirely 
masked.  On  the  other  hand,  if  too  little  copper  sulphate  is  used  a  light-colored 
precipitate  formed  by  uric  acid  and  purine  bases  may  obscure  the  brownish-red 
precipitate  of  cuprous  oxide.  The  action  of  KOH  or  NaOH  in  the  presence  of  an 
excess  of  sugar  and  insufficient  copper  will  produce  a  brownish  color.  Phosphates 
of  the  alkaline  earths  may  also  be  precipitated  in  the  alkaline  solution  and  be  mis- 
taken for  cuprous  hydroxide.     Trommer's  test  is  not  very  satisfactory. 

Salkowski^  has  proposed  a  modification  of  the  Trommer  procedure  which  he 
claims  is  a  very  accurate  sugar  test. 

(b)  Fehling's  Test. — To  about  i  c.c.  of  Fehling's  solution-  in  a  test-tube  add' 
about  4  CO.. of  water,  and  boil.^  [The  cupric  hydroxide  is  held  in  solution  by  the 
sodium  potassium  tartrate  (Rochelle  salt).]  This  is  done  to  determine  whether 
the  solution  will  of  itself  cause  the  formation  of  a  precipitate  of  brownish-red 
cuprous  oxide.  If  such  a  precipitate  forms,  the  Fehhng's  solution  must  not  be 
used.  Add  urine  to  the  hot  Fehhng's  solution,  a  few  drops  at  a  time,  and  heat  the 
mixture  to  boihng  after  each  addition  (never  add  more  urine  than  the  original 
volume  of  Fehling's  solution).  The  production  of  yellow  cuprous  hydroxide  or 
brownish-red  cuprous  oxide  indicates  that  reduction  has  taken  place.  The 
yellow  precipitate  is  more  hkely  to  occur  if  the  urine  is  added  rapidly  and  in  large 
amount,  whereas  with  a  less  rapid  addition  of  smaller  amounts  of  urine  the 
brownish-red  precipitate  is  generally  formed. 

This  is  a  much  more  satisfactory  test  than  Trommer's,  but  even 
this  test  is  not  entirely  reliable  when  used  to  detect  sugar  in  the  urine. 

'  Salkowski:  Zeit.  physiol.  Chem.,  79,  164,  191 2. 

-  Fehling's  solution  is  composed  of  two  definite  solutions — a  copper  sulphate  solution 
and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =  2,A-^S  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solution  =  1 25  grams  of  potassium  hydroxide  and  1 73  grams  of  Rochelle 
salt  dissoh-ed  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mixed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 

^  More  dilute  Fehling  solution  should  be  used  in  testing  urines  containing  small  amounts 
of  sugar.  In  case  of  urines  containing  a  high  concentration  of  sugar  it  may  sometimes  be 
desirable  to  use  a  larger  volume  of  Fehling's  solution. 


URINE  417 

Such  bodies  as  conjugate  glycuronates ,  uric  acid,  nucleo protein,  and  homo- 
gentisic  acid,  when  present  in  sufficient  amount,  may  produce  a  result 
similar  to  that  produced  by  sugar.  PJwsphates  of  the  alkaline  earths 
may  be  precipitated  by  the  alkali  of  the  Fehling's  solution  and  in  appear- 
ance may  be  mistaken  for  the  cuprous  hydroxide.  Cupric  hydroxide 
may  also  be  reduced  to  cuprous  oxide  and  this  in  turn  be  dissolved  by 
creatinine,  a  normal  urinary  constituent.  This  will  give  the  urine  under 
examination  a  greenish  tinge  and  may  obscure  the  sugar  reaction  even 
when  a  considerable  amount  of  sugar  is  present.  According  to  Laird^ 
even  small  amounts  of  creatinine  will  retard  the  reaction  velocity  of  re- 
ducing sugars  with  P'ehling's  solution. 

Conjugate  glycuronates  are  formed  after  the  ingestion  of  such  sub- 
stances as  chloral  hydrate,  camphor,  menthol,  thymol,  antipyrin, 
phenol,  etc.  The  chloral  hydrate  is  excreted  in  the  urine  as  trichlor- 
ethylglycuronic  acid,  C2CI3H2.C6H9O7.  This  compound  reduces  Fehl- 
ing's solution  and  is  levoiotdXory ,  whereas  glucose  also  reduces  but  is 
dextroioi2iiovy .  Therefore  by  means  of  a  polariscopic  test  we  may  dif- 
ferentiate between  a  "chloral  urine"  and  a  "sugar  urine." 

In  testing  urine  preserved  by  chloroform  a  positive  test  may  be  ob- 
tained in  the  absence  of  sugar.  This  is  due  to  the  fact  that  the  hot 
alkali  -produces  formic  acid  (a  reducing  fatty  acid)  from  the  chloroform. 

x^mmonium  salts  also  interfere  with  Fehling's  test.  If  present  in 
excess  the  urine  should  be  made  alkaHne  and  boiled  in  order  to  decom- 
pose the  ammonium  salts. 

(c)  Benedict's  Modifications  of  Fehling's  Test. — First  Modification. — To  2 
c.c.  of  Benedict's  solution-  in  a  test-tube  add  6  c.c.  of  distilled  water  and  7-^ 
drops  (not  more)  of  the  urine  under  examination.  Boil  the  mixture  vigorously 
for  about  15-30  seconds  and  permit  it  to  cool  to  room  temperature  spontaneously. 
(If  desired  this  process  may  be  repeated,  although  it  is  ordinarily  unnecessary.) 
If  sugar  is  present  in  the  solution  a  precipitate  will  form  which  is  often  bluish- 
green  or  green  at  first,  especially  if  the  percentage  of  sugar  is  low,  and  which 
usually  becomes  yellowish  upon  standing.  If  the  sugar  present  exceeds  0.06 
per  cent  this  precipitate  generally  forms  at  or  below  the  boiling-point,  whereas  if 
less  than  0.06  per  cent  of  sugar  is  present  the  precipitate  forms  more  slowly  and 
generally  only  after  the  solution  has  cooled.  The  greenish  precipitate  obtained 
with  urines  containing  small  amounts  of  sugar  may  be  a  compound  of  copper  with 
the  sugar  or  a  compound  of  some  constituent  of  the  urine  with  reduced  copper 

1  Laird:  Journ.  Path,  and  Bad.,  16,  398  191 2. 

*  Benedict's  modified  Fehling  solution  consists  of  two  definite  solutions — a  copper 
sulphate  solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =  34.65  grams  of  copper  sulphate  dissolved  in  water  and  made 
up  to  500  c.c. 

Alkaline  tartrate  solulion  =  ioo  grams  of  anhydrous  sodium  carbonate  and  173  grams  of 
Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered  bottles  and  mi.xed 
in  equal  volumes  when  needed  for  use.     This  is  done  to  prevent  deterioration. 
27 


41 8  PHYSIOLOGICAL   CHEMISTRY 

oxide  instead  of  being  a  precipitate  of  cuprous  hydroxide  or  oxide  as  is  the  case 
when  the  original  Fehhng  solution  is  reduced. 

Benedict  claims  that,  whereas  the  original  Fehling's  test  will  not 
serve  to  detect  sugar  when  present  in  a  concentration  of  less  than  o.i 
per  cent,  that  the  above  modification  will  serve  to  detect  sugar  when 
present  in  as  small  quantity  as  0.015-0.02  per  cent.  This  claim  has 
been  corroborated  by  Harrison.^  The  modified  solution  used  in  the 
above  test  differs  from  the  original  in  that  100  grams  of  sodium  car- 
bonate is  substituted  for  the  125  grams  of  potassium  hydroxide  ordi- 
narily used,  thus  forming  a  Fehhng  solution  which  is  considerably 
less  alkaline  than  the  original.  This  alteration  in  the  composition  of 
the  Fehling  solution  is  of  advantage  in  the  detection  of  sugar  in  the 
urine  inasmuch  as  the  strong  alkalinity  of  the  ordinary  Fehling  solu- 
tion has  a  tendency,  when  the  reagent  is  boiled  with  a  urine  containing 
a  small  amount  of  glucose,  to  decompose  sufficient  of  the  sugar  to 
render  the  detection  of  the  remaining  portion  exceedingly  difficult 
by  the  usual  technic.  Benedict  claims  that  for  this  reason  the  use  of 
his  modified  solution  permits  the  detection  of  smaller  amounts  of  sugar 
than  does  the  use  of  the  ordinary  Fehling  solution. 

Second  Modification.  ^ — ^Benedict  has  fitrther  modified  his  solution  and  has 
succeeded  in  obtaining  one  which  does  not  deteriorate  upon  long  standing.* 
The  following  is  the  procedure  for  the  detection  of  glucose  in  the  urine :  To  5  c.c. 
of  the  reagent  in  a  test-tube  add  8  (not  more)  drops  of  the  urine  to  be  examined. 
The  fluid  is  then  boiled  vigorously  for  from  one  to  two  minutes  and  then  allowed 
to  cool  spontaneously.  In  the  presence  of  glucose  the  entire  body  of  the  solution 
will  be  filled  with  a  precipitate,  which  may  be  red,  yellow,  or  green  in  color,  de- 
pending upon  the  amount  of  sugar  present.  If  no  glucose  is  present,  the  solution 
will  either  remain  perfectly  clear,  or  will  show  a  very  faint  turbidity,  due  to 
precipitated  urates. 

Even  very  small  quantities  ot  glucose  in  urine  (o.i  per  cent) 
yield  precipitates  of  surprising  bulk  with  this  reagent,  and  the  positive 
reaction  for  glucose  is  the  filhng  of  the  entire  body  of  the  solution 
with  a  precipitate,  so  that  the  solution  becomes  opaque.     Since  amount 

1  Harrison:  Pharm.  Jour.,  87,  746,  191 1. 

2  Benedict:  Jour.  Am.  Med.  Ass'n,  57,  1193,  191X. 
^Benedict's  new  solution  has  the  following  composition: 

Copper  sulphate 17  •  3  g™- 

Sodium  citrate 1 73 .  o  gm. 

Sodium  carbonate  (anhydrous) 100. o  gm. 

Distilled  water  to 1000. o  c.c. 

With  th'e  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in  about  600  c.c.  of  water. 
Pour  (through  a  folded  filter  if  necessary)  into  a  glass  graduate  and  make  up  to  850  c.c. 
Dissolve  the  copper  sulphate  in  about  100  c.c.  of  water  and  make  up  to  150  c.c.  Pour  the 
carbonate-citrate  solution  into  a  large  beaker  or  casserole  and  add  the  copper  sulphate 
solution  slowly,  with  constant  stirring.  The  mixed  solution  is  ready  for  use,  and  does  not 
deteriorate  upon  long  standing. 


URINE  419 

rather  than  color  of  the  precipitate  is  made  the  basis  of  this  test,  it 
may  be  applied,  even  for  the  detection  of  small  quantities  of  glucose, 
as  readily  in  artificial  light  as  in  daylight.  Chloroform  does  not  in- 
terfere with  this  test  nor  do  uric  acid  or  creatinine  interfere  to  such 
an  extent  as  in  the  case  of  Fehling's  test. 

(d)  Haines'  Test. — This  is  a  copper  reduction  test  similar  in  many 
respects  to  the  Fehling  and  Benedict  reactions.  In  Haines'  solution^ 
the  cupric  hydroxide  is  held  in  solution  by  glycerol  instead  of  Rochelle 
salt  as  in  Fehling's  solution. 

Perform  the  test  as  follows :  Introduce  about  5  c.c.  of  Haines'  solution^  into  a 
test-tube  and  heat  to  boUing.  If  no  reduction  occurs  add  6-8  drops  of  the  urine 
and  again  bring  to  a  boil.  If  glucose  is  present  an  abundant  yeUow  ("cuprous 
hydroxide)  or  brownish-red  ("cuprous  oxidej  precipitate  is  thrown  down.  This 
test  is  about  as  delicate  as  Fehling's  test. 

(e)  Allen's  Modification  of  Fehling's  Test. — The  following  procedure  is  recom- 
mended: "From  7  to  8  c.c.  of  the  sample  of  urine  to  be  tested  is  heated  to  boiling 
in  a  test-tube,  and,  without  separating  any  precipitate  of  albumin  which  may  be  pro- 
duced, 5  c.c.  of  the  solution  of  copper  sulphate  used  for  preparing  Fehling's  solution  is 
added.  This  produces  a  precipitate  containing  uric  acid,  xanthine,  hj-poxanthine, 
phosphates,  etc.  To  render  the  precipitation  complete,  however,  it  is  desirable 
to  add  to  the  liquid,  when  partially  cooled,  from  i  to  2  c.c.  of  a  saturated  solution  of 
sodium  acetate  having  a  feebly  acid  reaction  to  litmus. ^  The  liquid  is  filtered  and 
to  the  filtrate,  which  will  have  a  bluish-green  color,  5  c.c.  of  the  alkaline  tartrate 
mixture  used  for  preparing  Fehling's  solution  is  added,  and  the  liquid  boiled  for 
15-20  seconds.  In  the  presence  of  more  than  0.25  per  cent  of  sugar,  separation  of 
cuprous  oxide  occurs  before  the  boUing-point  is  reached;  but  with  smaller  quantities 
precipitation  takes  place  during  the  cooling  of  the  solution,  which  becomes  greenish, 
opaque,  and  suddenly  deposits  cuprous  oxide  as  a  fine  brownish-red  precipitate." 

Mercuric  Oxide  Reduction  Test  (Cramer).^ — This  test  depends  on 
the  reduction  of  mercuric  oxide  in  a  weakly  alkaline  solution  with  the 
formation  of  metallic  mercury.  The  degree  of  alkalinity  is  an  impor- 
tant factor,  as  the  test  becomes  more  sensitive  but  less  specific  tke 
greater  the  alkalinity  of  the  reagent. 

Apply  the  test  as  follows : 

Introduce  3  c.c.  of  Cramer's  "2.5  standard  reagent"*  into  a  test-tube  and  heat 
to  boiling.  The  reagent  remains  clear  but  becomes  shghtly  yellow.  Add  3  c.c. 
of  urine  and  heat  the  mixture  to  boihng.     Remove  the  tube  from  the  flame,  and 

1  Haines  solution  may  be  prepared  by  dissolving  8.314  grams  of  copper  sulphate  in 
400  c.c.  of  water  adding  40  c.c.  of  glycerol  and  500  c.c.  of  5  per  cent  potassium  hydroxide 
solution. 

*  Sufficient  acetic  acid  should  be  added  to  the  sodium  acetate  solution  to  render  it  feebly 
acid  to  litmus.  A  saturated  solution  of  sodium  acetate  keeps  well,  but  weaker  solutions  are 
apt  to  become  mouldy,  and  then  possess  the  power  of  reducing  Fehling's  solution.  Hence 
it  is  essential  in  all  cases  of  importance  to  make  a  blank  test  by  mixing  equal  measures  of 
copper  sulphate  solution,  alkahne  tartrate  solution  and  water,  adding  a  little  sodium  acetate 
solution,  and  heating  the  mixture  to  boiling. 

'Cramer:  Bioch.  Jour.,  9,  156,  1915. 

*  See  Chapter  II,  page  28. 


420  PHYSIOLOGICAL    CHEMISTRY 

after  30  seconds  acidify  with  a  few  drops  of  acetic  acid.  Hold  the  tube  over 
ordinary  print.  If  the  tirine  is  normal,  a  slight  but  distinct  turbidity  remains 
but  the  print  is  clearly  readable.  If  sugar  is  present  in  the  urine  above  the  nor- 
mal concentration,  the  contents  of  the  tube  darken  and  on  standing  a  finely  di- 
vided precipitate  of  mercury  settles  to  the  bottom  of  the  tube.  The  amount  of 
the  precipitate  depends  upon  the  concentration  of  reducing  sugar  in  the  urine. 

It  is  claimed  that  this  test  is  free  from  fallacies  inherent  in  Fehling's 
test  as  the  result  of  the  reducing  action  of  uric  acid  and  creatinine. 
The  test  is  said  to  be  more  sensitive  than  Fehling's  test  or  the  bismuth 
reduction  tests,  and  is  particularly  suitable  for  the  examination  of 
urines  in  which  the  amount  of  sugar  present  exceeds  the  normal  amount 
only  slightly. 

If  the  reagent  be  made  more  alkaline  than  indicated,  it  ceases  to  be 
specific  for  reducing  sugars. 

(f)  Bismuth  Reduction  Test  (Boettger). — To  5  c.c.  of  urine  in  a  test-tube  add 
I  c.c.  of  KOH  or  NaOH  and  a  very  small  amount  of  bismuth  subnitrate,  and  boil. 
The  solution  will  gradually  darken  and  finally  assume  a  black  color  due  to  reduced 
bismuth.  If  the  test  is  made  with  urine  containing  albumin  this  must  be  removed, 
by  boiling  and  filtering,  before  applying  the  test,  since  with  albumin  a  similar  change 
of  color  is  produced  (bismuth  sulphide). 

(g)  Bismuth  Reduction  Test  (Nylander). — To  5  c.c.  of  urine  in  a  test-tube 
add  one-tenth  its  volume  of  Nylander's  reagent^  and  heat  for  five  minutes 
in  a  boiling  water-bath.  ^  The  mixture  will  darken  if  reducing  sugar  is  present 
and  upon  standing  for  a  few  moments  a  black  color  will  appear. 

This  color  is  due  to  the  precipitation  of  bismuth.  If  the  test  is 
made  on  urine  containing  albumin  this  must  be  removed,  by  boiling 
and  filtering,  before  applying  the  test,  since  with  albumin  a  similar 
change  of  color  is  produced.  Glucose  when  present  to  the  extent  of 
0.08  per  cent  may  be  easily  detected  by  this  reaction  (Rabe^  claims  that 
o.oi  per  cent  may  be  so  detected).  Uric  acid  and  creatinine  which 
interfere  with  the  Fehling  test  do  not  interfere  with  the  Nylander's 
reaction.  It  is  claimed  by  Bechold  that  the  bismuth  reduction  tests 
give  a  negative  reaction  with  solutions  containing  sugar  when  mercuric 
chloride  or  chloroform  is  present.  Other  observers^  have  failed  to 
verify  the  inhibitory  action  of  the  mercuric  chloride  and  have  shown 
that  the  inhibitory  influence  of  chloroform  may  be  overcome  by  raising 

1  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth  subnitrate  and  4  grams 
of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  potassium  hydro.xide  solution.  The  reagent  is 
then  cooled  and  filtered. 

^Hammarsten  suggests  that  the  solution  be  boiled  for  2-5  minutes  (according  to  the 
sugar  content)  over  a  free  flame  and  the  tube  then  permitted  to  stand  five  minutes  before 
drawing  conclusions. 

*Rabe:  Apolh.  Ztg.,  29,  554,  1914. 

*  Rehfuss  and  Hawk:  Jour.  Biol.  Chem.,  7,  267,  1910;  also  Zeidlitz:  Upsala  Lakdre- 
foren  Fork,  N.  F.,  11,  1906. 


URINE  421 

the  temperature  of  the  urine  to  the  boiling-point  for  a  period  of  five 
minutes  previous  to  making  the  test. 

Urines  rich  in  indican,  uroerythrin,  urochrome  or  Jiema  to  porphyrin, 
as  well  as  urines  excreted  after  the  ingestion  of  large  amounts  of  certain 
medicinal  substances,  may  give  a  darkening  of  the  Xylander's  reagent 
similar  to  that  of  a  true  sugar  reaction.  It  is  a  disputed  point  whether 
the  urine  after  the  administration  of  urotropin  will  reduce  the  Xylan- 
der  reagent.^ 

Strausz-  has  recently  shown  that  the  urine  of  diabetics  to  whom 
"lothion"  (diiodohydroxypropane)  has  been  administered  will  give  a 
negative  Nylander's  reaction  and  respond  positively  to  the  Fehling  and 
polarization  tests.  "lothion"  also  interferes  with  the  Nylander  test 
in  vitro  whereas  KI  and  I  do  not. 

According  to  Rustin  and  Otto  the  addition  of  PtCU  increases  the 
delicacy  of  Nylander's  reaction.  They  claim  that  this  procedure  causes 
the  sugar  to  be  converted  quantitatively.  No  quantitative  method  has 
yet  been  devised,  however,  based  upon  this  principle. 

A  positive  bismuth  reduction  test  is  probably  due  to  the  following 
reactions: 

(a)  Bi(OH)2(NO)3+KOH->Bi(OH)3+KN03. 

(b)  2Bi(OH)3— 30-^Bi2+3H20. 

Bohmansson,^  before  testing  the  urine  under  examination  treats  it 
(10  c.c.)  with  1 5  volume  of  25  per  cent  hydrochloric  acid  and  }i  volume 
of  boneblack.  This  mixture  is  shaken  one  minute,  then  filtered, 
and  the  neutralized  filtrate  tested  by  Nylander's  reaction.  Bohmansson 
claims  that  this  procedure  removes  certain  interfering  substances, 
notably  urochrome. 

6.  Fermentation  Test. — Rub  up  in  a  mortar  about  15  c.c.  of  the  urine  with  a 
small  piece  of  compressed  yeast.  Transfer  the  mixture  to  a  saccharometer 
(Fig-  3>  page  31 )  and  stand  it  aside  in  a  warm  place  for  about  12  hours.  If  glu- 
cose is  present,  alcohoUc  fermentation  will  occur  and  carbon  dioxide  will  collect 
as  a  gas  in  the  upper  portion  of  the  tube.  On  the  completion  of  fermentation, 
introduce,  by  means  of  a  bent  pipette,  a  little  KOH  solution  into  the  graduated 
portion,  place  the  thumb  tightly  over  the  opening  in  the  apparatus  and  invert  the 
saccharometer.  Remembering  that  KOH  has  the  power  to  absorb  COj  how 
do  you  explain  the  result?' 

7.  Polariscopic  Examination. — For  directions  as  to  the  use  of  the  polariscope 
see  Chapter  II. 

'  Abt:  Archives  of  Pediatrics,  24,  275,  1907;  also  VVeitbrecht:  Schweiz.  Woch.,  47,  577, 
1909. 

"Strausz:  Miiiich.  mcd.  Woch.,  59,  85,  1912. 

^Bohmansson:  Biochcm.  ZciL,  19,  p.  281. 

*  The  findings  of  Neuberg  and  associates  indicate  that  the  liberation  of  carbon  dioxide 
by  yeast  is  not  necessarily  a  criterion  of  the  presence  of  sugar.  The  presence  of  an  enzyme 
called  carboxylase  has  been  demonstrated  in  yeast  which  has  the  power  of  splitting  off  COi 
from  the  carboxyl  group  of  amino-  and  other  aliphatic  acids. 


42  2  PHYSIOLOGICAL   CHEMISTRY 

PROTEINS 

Normal  urine  contains  a  trace  of  protein  material,  but  the  amount 
present  is  so  slight  as  to  escape  detection  by  any  of  the  simple  tests  in 
general  use  for  the  detection  of  protein  urinary  constituents.  The 
following  are  the  more  important  forms  of  protein  material  which  have 
been  detected  in  the  urine  under  pathological  conditions: 

(i)  Serum  albumin. 

(2)  Serum  globulin. 

I    Deutero-proteose. 

(3)  Proteoses      \    Hetero-proteose. 

[    "Bence- Jones'  protein." 

(4)  Peptone. 

(5)  Nucleoprotein. 

(6)  Fibrin. 

(7)  Oxyhemoglobin. 

ALBUMIN 

Normal  urine  contains  a  trace  of  albumin  which  is  too  slight  to  be 
detected  by  the  usual  procedures. 

Albuminuria  is  a  condition  in  which  serum  albumin  or  serum  globulin 
appears  in  the  urine.  There  are  two  distinct  forms  of  albuminuria,  i.e., 
renal  albuminuria  and  accidental  albuminuria.  Sometimes  the  terms 
"true"  albuminuria  and  "false"  albuminuria  are  substituted  for  those 
just  given.  In  the  renal  type  the  albumin  is  excreted  by  the  kidneys. 
This  is  the  more  serious  form  of  the  malady  and  at  the  same  time  is  more 
frequently  encountered  than  the  accidental  type.  Among  the  causes  of 
renal  albuminuria  are  altered  blood  pressure  in  the  kidneys,  altered 
kidney  structure,  or  changes  in  the  composition  of  the  blood  entering 
the  kidneys,  thus  allowing  the  albumin  to  diffuse  more  readily.  In  the 
accidental  form  of  albuminuria  the  albumin  is  not  excreted  by  the 
kidneys  as  is  the  case  in  the  renal  form  of  the  disorder,  but  arises  from 
the  blood,  lymph,  or  some  albumin-containing  exudate  coming  into 
contact  with  the  urine  at  some  point  below  the  kidneys.  It  has  been 
suggested^  that  albuminurias  may  be  classed  as  pre-renal,  renal  and 
post-renal.  The  pre-renal  type  is  illustrated  by  the  albuminuria  of 
heart  disease,  whereas  the  post-renal  form  corresponds  to  what  we  have 
called  "accidental"  albuminuria. 

The  determination  of  albumin  may  be  of  assistance  in  following  the 
course  of  kidney  disturbances,  but  the  results  can  only  be  interpreted 
in  the  light  of  other  clinical  findings. 

^  Bruce:  Lancet,  May  6,  191 1,  p.  1205. 


URINE  423 

Experiments 
(The  urine  should  be  filtered  before  performing  these  tests.) 

Nitric  Acid  Ring  Test  (Heller.) — Place  5  c.c.  of  concentrated  HNO3  in  a  test- 
tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the  urine  to  flow  slowly 
down  the  side.  The  liquids  should  stratify  with  the  formation  of  a  white  zone 
of  precipitated  albumin  at  the  point  of  juncture. 

If  the  albumin  is  present  in  very  small  amount  the  white  zone  may 
not  form  until  the  tube  has  been  allowed  to  stand  for  several  minutes. 
If  the  urine  is  quite  concentrated  a  white  zone,  due  to  uric  acid  or  urates, 
will  form  upon  treatment  with  nitric  acid  as  indicated.  This  ring  may 
be  easily  ditlerentiated  from  the  albumin  ring  by  repeating  the  test 
after  diluting  the  urine  with  3  or  4  volumes  of  water,  whereupon  the 
ring,  if  due  to  uric  acid  or  urates,  will  not  appear.  It  is  ordinarily 
possible  to  differentiate  between  the  albumin  ring  and  the  uric  acid  ring 
without  diluting  the  urine,  since  the  ring,  when  due  to  uric  acid,  has 
ordinarily  a  less  sharply  defined  upper  border,  is  generally  broader  than 
the  albumin  ring  and  frequently  is  situated  in  the  urine  above  the  point 
of  contact  with  the  nitric  acid.  Concentrated  urines  also  occasionally 
exhibit  the  formation,  at  the  point  of  contact,  of  a  crystalline  ring  with 
very  sharply  defined  borders.  This  is  urea  nitrate  and  is  easily  dis- 
tinguished from  the  "fluffy"  ring  of  albumin.  If  there  is  any  diflBi- 
culty  in  differentiation  a  simple  dilution  of  the  urine  with  water,  as 
above  described,  will  remove  the  difficulty.  Various  colored  zones,  due 
either  to  the  presence  of  indican,  bile  pigments,  or  to  the  oxidation  of 
other  organic  urinary  constituents,  may  form  in  this  test  under  certain 
conditions.  These  colored  rings  should  never  be  confounded  with  the 
white  ring  which  alone  denotes  the  presence  of  albumin. 

After  the  administration  of  certain  drugs  a  white  precipitate  of 
resin  acids  may  form  at  the  point,  of  contact  of  the  two  fluids  and  may 
cause  the  observer  to  draw  wrong  conclusions.  This  ring,  if  composed 
of  resin  acids,  will  dissolve  in  alcohol,  whereas  the  albumin  ring  will  not 
dissolve  in  this  solvent. 

Weinberger  has  shown  that  a  ring  closely  resembling  the  albumin 
ring  is  often  obtained  in  urines  preserved  for  a  considerable  time  by 
thymol  when  subjected  to  the  nitric  acid  test.  The  ring  is  due  to  the 
formation  of  nitrosothymol  and  possibly  nitrothymol.  If  the  thymol 
is  removed  from  the  urine  by  extraction  with  petroleum  ether'  previous 
to  adding  nitric  acid,  the  ring  does  not  form. 

An  instrument  called  the  albumoscope  ijiorismascope)  has  been  de- 

1  Accomplished  readily  by  gently  agitating  equal  volumes  of  petroleum  ether  and  the 
urine  under  examination  for  two  minutes  in  a  test-tube  before  applying  the  test. 


424 


PHYSIOLOGICAL   CHEMISTRY 


vised  for  use  in  this  test  and  has  met  with  considerable  favor.     The 
method  of  using  the  albumoscope  is  described  below. 

Use  of  the  Albumoscope. — This  instrument  is  intended  to  facilitate 
the  making  of  "ring"  tests  such  as  Heller's  and  Roberts'.  In  making 
a  test  about  5  c.c.  of  the  solution  under  examination  is  first  intro- 
duced into  the  apparatus  through  the  larger  arm  (see  Fig.  130), 
and  the  reagent  used  in  the  particular  test  is  then  introduced  through 
the  capillary  arm  and  allowed  to  flow  down  underneath  the  solution 
under  examination.  If  a  reasonable  amount  of  care  is  taken  there  is 
no  possibility  of  mixing  the  two  solutions  and  a  defi- 
nitely defined  white  "ring"  is  easily  obtained  at  the 
zone  of  contact. 

2.  Nitric  Acid  and  Magnesium  Sulphate  Ring  Test 
(Roberts). — ^Place  5  c.c.  of  Roberts'  reagent^  in  a  test- 
tube,  incline  the  tube,  and  by  means  of  a  pipette  allow  the 
urine  to  flow  slowly  down  the  side.  The  liquids  shovild 
stratify  with  the  formation  of  a  white  zone  of  precipitated 
albumin  at  the  point  of  juncture. 

This  test  is  a  modification  of  Heller's  ring  test 
and  is  rather  more  satisfactory  than  that  test,  since 
the  colored  rings  never  form  and  the  consequent 
confusion  is  avoided.  The  albumoscope  (see  above) 
may  also  be  used  in  making  this  test. 


Fig. 


130. — Albumo- 
scope. 


3.  Spiegler's  Ring  Test. — Place  5  c.c.  of  Spiegler's  reagent^  in  a  test-tube,  in- 
cline the  tube  and,  by  means  of  a  pipette,  allow  5  c.c.  of  urine,  acidified  with  acetic 
acid,  to  flow  slowly  down  the  side.  A  white  zone  wUl  form  at  the  point  of  contact. 
This  is  an  exceedingly  delicate  test,  in  fact  too  delicate  for  ordinary  clinical  purposes, 
since  it  serves  to  detect  albumin  when  present  in  the  merest  trace  (i  :  250,000)  and 
hence  most  normal  urines  wUl  give  a  positive  reaction  for  albumin  when  this  test  is 
applied.     Proteose  and  peptone  are  also  said  to  respond  to  this  test. 

4.  Coagulation  or  Boiling  Test. — (a)  Heat  5  c.c.  of  urine  to  boiling  in  a  test- 
tube.  (K  the  urine  is  not  clear  it  should  be  filtered.)  A  precipitate  forming  at 
this  point  is  due  either  to  albumin  or  to  phosphates.  Acidify  the  urine  slightly 
by  the  addition  of  3-5  drops  of  very  dilute  acetic  acid,  adding  the  acid  drop  by 
drop  to  the  hot  solution.  If  the  precipitate  is  due  to  phosphates  it  will  disappear 
under  these  conditions,  whereas  if  it  is  due  to  albumin  it  will  not  only  fail  to 
disappear  but  will  become  more  fiocculent  in  character,  since  the  reaction  of  a 

1  Robert's  reagent  is  composed  of  i  volume  of  concentrated  HNO3  and  5  volumes  of  a 
saturated  solution  of  MgSO*. 

*  Spiegler's  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

Mercuric  chloride 40  grams. 

Sodium  chloride 5°  grams. 

Glycerol 100  grams. 

Distilled  water 1000  grams. 


URINE  425 

fluid  must  be  acid  to  secure  the  complete  precipitation  of  the  albumin  by  this 
coagulation    process. 

Too  much  acid  should  be  avoided  since  it  will  cause  the  albumin  to 
go  into  solution.  Certain  resin  acids  may  be  precipitated  by  the 
acid,  but  the  precipitate  due  to  this  cause  may  be  easily  differentiated 
from  the  albumin  precipitate  by  reason  of  its  solubility  in  alcohol. 

(b)  A  modification  of  this  test  in  quite  general  use  is  as  follows :  Fill  a  test- 
tube  two-thirds  full  of  urine  and  gently  heat  the  upper  half  of  the  fluid  to  boiling, 
being  careful  that  this  fluid  does  not  mix  with  the  lower  half.  A  turbidity  indi- 
cates albumin  or  phosphates.  Acidify  the  urine  sUghtly  by  the  addition  of  3-5 
drops  of  dilute  acetic  acid,  when  the  turbidity,  if  due  to  phosphates,  will  disappear. 

Nitric  acid  is  often  used  in  place  of  acetic  acid  in  these  tests.  In 
case  nitric  acid  is  used  ordinarily  1-2  drops  is  sufficient. 

5.  Acetic  Acid  and  Potassium  Ferrocyanide  Test. — To  5  c.c.  of  urine  in  a 
test-tube  add  5-10  drops  of  acetic  acid.  Mix  well  and  add  potassium  ferro- 
cyanide drop  by  drop,  until  a  precipitate  forms. 

This  is  a  very  delicate  test.  Schmiedl  claims  that  a  precipitate  of 
Fe(Cn)6K2Zn  or  Fe(Cn)6Zn2  is  formed  when  urines  containing  zinc 
are  subjected  to  this  test  and  that  this  precipitate  resembles  the 
precipitate  secured  with  protein  solutions.  In  the  case  of  human 
urine  a  reaction  was  obtained  when  0.000022  gram  of  zinc  per  cubic 
centimeter  was  present.  Schmiedl  further  found  that  the  urine  col- 
lected from  rabbits  housed  in  zinc-lined  cages  possessed  a  zinc  content 
which  was  sufficient  to  yield  a  ready  response  to  the  test. 

Proteoses  may  also  be  detected  by  this  test.  To  differentiate 
albumin  from  proteose  perform  the  coagulation  test  (see  page  424). 

6.  Tanret's  Test. — To  5  c.c.  of  urine  in  a  test-tube  add  Tanret's  reagent*  drop 
by  drop  until  a  turbidity  or  precipitate  forms.  This  is  an  exceedingly  delicate  test. 
Sometimes  the  urine  is  stratified  upon  the  reagent  as  in  Heller's  or  Roberts'  ring 
test.  According  to  Repiton,  urates  interfere  with  the  delicacy  of  this  test.  Tanret, 
however,  claims  that  urates  do  not  interfere  inasmuch  as  any  precipitate  due  to 
urates  may  be  brought  into  solution  by  heat,  whereas  an  albumin  precipitate  under 
the  same  conditions  will  persist.  Tanret  further  states  that  mucin  interferes  with 
the  delicacy  of  the  test  and  that  it  should  therefore  be  removed  from  the  urine 
under  examination  by  acidification  with  acetic  acid  and  filtration  before  testing  for 
albumin.     This  test  also  serves  to  detect  proteoses. 

7.  Sodium  Chloride  and  Acetic  Acid  Test, — Mix  two  volumes  of  urine  and  one 
volume  of  a  saturated  solution  of  sodium  chloride  in  a  test-tube,  acidify  with  acetic 
acid,  and  heat  to  boiling.  The  production  of  a  cloudiness  or  the  formation  of  a 
precipitate  indicates  the  presence  of  albumin.     The  resin  acids  may  interfere  here 

^Tanret's  reagent  is  prepared  as  follows:  Dissolve  1.35  grams  of  mercuric  chloride  in 
25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide  dissolved  in  25  c.c.  of 
water,  then  make  the  total  solution  up  to  60  c.c.  with  water  and  add  20  c.c.  of  glacial  acetic 
add  to  the  mixture. 


426  PHYSIOLOGICAL   CHEMISTRY 

as  in  the  ordinary  coagulation  test  (page  424),  but  they  may  be  easily  differentiated 
from  albumin  by  means  of  their  solubility  in  alcohol. 

GLOBULIN 

Serum  globulin  is  not  a  constituent  of  normal  urine  but  frequently 
occurs  in  the  urine  under  pathological  conditions  and  is  ordinarily 
associated  with  serum  albumin.  In  albuminuria  globulin  in  varying 
amounts  often  accompanies  the  albumin,  and  the  clinical  significance 
of  the  two  is  very  similar.  Under  certain  conditions  globulin  may  occur 
in  the  urine  unaccompanied  by  albumin. 

Experiments 

Globulin  will  respond  to  all  the  tests  just  outlined  under  Albumin. 
If  it  is  desirable  to  differentiate  between  albumin  and  globulin  in  any 
urine  the  following  processes  may  be  employed: 

1.  Saturation  with  Magnesium  Sulphate. — Place  25  c.c.  of  neutral  urine  in 
a  small  beaker  and  add  pulverized  magnesium  sulphate  in  substance  to  the  point 
of  saturation.  If  the  protein  present  is  globulin  it  will  precipitate  at  this  point. 
If  no  precipitate  is  produced  acidify  the  saturated  solution  with  acetic  acid  and 
warm  gently.     Albumin  will  be  precipitated  if  present. 

The  above  procedure  may  be  used  to  separate  globulin  and  albumin 
if  present  in  the  same  urine.  To  do  this  filter  off  the  globulin  after  it 
has  been  precipitated  by  the  magnesium  sulphate,  then  acidify  the  clear 
solution  and  warm  gently  as  directed.  Note  the  formation  of  the 
albumin  precipitate. 

2.  Half-saturation  with  Ammonimn  Sulphate. — Place  25  c.c.  of  neutral  urine 
in  a  small  beaker  and  add  an  equal  volume  of  a  saturated  solution  of  ammonium 
sulphate.  Globulin,  if  present,  will  be  precipitated.  If  no  precipitate  forms  add 
ammonium  sulphate  in  substance  to  the  point  of  saturation.  If  albumin  is  present 
it  will  be  precipitated  upon  saturation  of  the  solution  as  just  indicated.  This 
method  may  also  be  used  to  separate  globulin  and  albumin  when  they  occur  in  the 
same  urine. 

Frequently  in  urine  which  contains  a  large  amount  of  urates  a  precipitate  of 
ammonium  urate  may  occur  when  the  ammonium  sulphate  solution  is  added  to  the 
urine.  This  urate  precipitate  should  not  be  confounded  with  the  precipitate  due 
to  globulin.  The  two  precipitates  may  be  differentiated  by  means  of  the  fact  that 
the  urate  precipitate  ordinarily  appears  only  after  the  lapse  of  several  minutes 
whereas  the  globulin  generally  precipitates  at  once. 

PROTEOSE  AND  PEPTONE 

Proteoses,  particularly  deutero-proteose  and  hetero-proteose,  have 
frequently  been  found  in  the  urine  under  various  pathological  con- 
ditions such  as  diphtheria,   pneumonia,  intestinal  ulcer,   carcinoma, 


URINE  427 

dermatitis,  osteomalacia,  atrophy  of  the  kidneys,  and  in  sarcomata 
of  the  bones  of  the  trunk.  The  presence  of  proteose  in  the  urine  may 
frequently  be  demonstrated  in  any  pathological  condition  in  which  there 
is  absorption  of  partially  digested  pus.  "Bence-Jones'  protein,"  a 
proteose-like  substance,  is  of  interest  in  this  connection  and  its  appear- 
ance in  the  urine  is  believed  to  be  of  great  diagnostic  importance  in 
cases  of  multiple  myeloma  or  myelogenic  osteosarcoma.  By  some  in- 
vestigators this  protein  is  held  to  be  a  variety  of  hetero-proteose,  whereas 
others  claim  that  it  possesses  albumin  characteristics.  The  origin  of 
"Bence- Jones'  protein"  is  unknown.  Its  origin  has  at  various  times 
been  ascribed  to  the  blood  proteins,  the  bones  or  to  abnormal  metabo- 
lism of  protein  material  in  the  body.  It  occurs  in  the  urine  in  about 
80  per  cent  of  the  cases  of  multiple  myeloma.  If  its  presence  is  unac- 
companied by  multiple  myeloma  it  is  nearly  always  associated  with 
some  disease  of  the  blood-forming  organs  or  of  the  bones.  When 
"Bence-Jones'  protein"  is  hydrolyzed  it  is  found  to  contain  all  the  amino- 
acids  which  are  characteristic  of  typical  proteins. 

Peptone  certainly  occurs  much  less  frequently  as  a  constituent  of 
the  urine  than  does  proteose,  in  fact  most  investigators  seriously  ques- 
tion its  presence  under  any  conditions.  .  There  are  many  instances 
of  peptonuria  cited  in  the  early  literature,  but  because  of  the  uncertainty 
in  the  conception  of  what  really  constituted  a  peptone  it  is  probable  that 
in  many  cases  of  so-called  peptonuria  the  protein  present  was  really 
proteose. 

Experiments 

I.  Phosphotungstic  Precipitation  Test  (v.  Aldor). — ^Acidify  10  c.c.  of  urine 
with  hydrochloric  acid,  add  phosphotungstic  acid  until  no  more  precipitate  forms 
and  centrifugate'  the  solution.  Decant  the  supernatant  fluid,  add  some  abso- 
lute alcohol  to  the  precipitate,  and  centrifugate  again.  This  washing  with  alcohol 
is  intended  to  remove  the  urobilin  and  hence  should  be  continued  so  long  as  the 
alcohol  exhibits  any  coloration  whatever.  Now  suspend  the  precipitate  in  water 
and  add  potassium  hydroxide  to  bring  it  into  solution.  At  this  point  the  solution 
may  be  blue  in  color,  in  which  case  decolorization  may  be  secured  by  gently 
heating.  Apply  the  biuret  test  to  the  cool  solution.  A  positive  biuret  test  indi- 
cates the  presence  of  proteoses. 

2.  Boiling  Test. — Make  the  ordinary  coagulation  test  according  to  the  di- 
rection given  under  Albumin,  page  424.  If  no  coagulable  protein  is  found  allow 
the  boiled  urine  to  stand  and  note  the  gradual  appearance,  in  the  cooled  fluid,  of 
a  flaky  precipitate  of  proteose.  Spiegler's  reaction  may  also  be  applied  at  this 
point.     A  precipitalc  indicates  proteose. 

3.  Schulte's  Method. — Acidify  50  c.c.  of  urine  with  dilute  acetic  acid  and  filter 
off  any  precipitate  of  nucleoprotein  which  may  form.     Now  test  a  few  cubic  centi- 

^  If  not  convenient  to  use  a  centrifuge  the  precipitate  may  be  filtered  off  and  washed  on 
the  filter  paper  with  alcohol. 


428  PHYSIOLOGICAL    CHEMISTRY 

meters  of  the  urine  for  coagulable  protein,  by  tests  2  and  4  under  Albumin,  pages 
424-5.  If  coagulable  protein  is  present  remove  it  by  coagulation  and  filtration 
before  proceeding.  Introduce  25  c.c.  of  the  urine,  freed  from  coagulable  protein, 
into  150  c.c.  of  absolute  alcohol  and  allow  it  to  stand  for  12-24  hours.  Decant  the 
supernatant  fluid  and  dissolve  the  precipitate  in  a  small  amount  of  hot  water.  Now 
filter  this  solution,  and  after  testing  again  for  nucleoprotein  with  very  dilute  acetic 
acid,  try  the  biuret  test.     If  this  test  is  positive  the  presence  of  proteose  is  indicated.^ 

Urobilin  does  not  ordinarily  interfere  with  this  test  since  it  is  almost  entirely 
dissolved  by  the  absolute  alcohol  when  the  proteose  is  precipitated. 

4.  Detection  of  "Bence-Jones'  Protein." — Heat  the  suspected  urine  very 
gently,  carefully  noting  the  temperature.  At  as  low  a  temperature  as  4o°C.  a 
turbidity  may  be  observed,  and  as  the  temperature  is  raised  to  about  6o°C.  a 
floccvdent  precipitate  forms  and  clings  to  the  sides  of  the  test-tube.  If  the  urine 
is  now  acidified  very  slightly  with  acetic  acid  and  the  temperature  further  raised 
to  ioo°C.  the  precipitate  at  least  partly  disappears;  it  wiU  return  upon  cooling 
the  tube. 

This  property  of  precipitating  at  so  low  a  temperature  and  of  dissolving  at  a 
higher  temperature  is  typical  of  "Bence-Jones'  protein"  and  may  be  used  to  differ- 
entiate it  from  all  other  forms  of  protein  material  occurring  in  the  urine. 

NUCLEOPROTEIN 

There  has  been  considerable  controversy  as  to  the  proper  classifica- 
tion for  the  protein  body  which  forms  the  "nubecula"  of  normal  urine. 
By  different  investigators  it  has  been  called  mucin,  mucoid,  phospho- 
protein,  nucleoalhumin,  and  nucleoprotein.  Of  course,  according  to 
the  modern  acceptation  of  the  meanings  of  these  terms  they  cannot  be 
synonymous.  Mucin  and  mucoid  are  glycoproteins  and  hence  contain 
no  phosphorus  (see  page  112),  whereas  phosphoproteins  and  nucleo- 
proteins  are  phosphorized  bodies.  It  may  possibly  be  that  both  these 
forms  of  protein,  i.e.,  the  glycoprotein  and  the  phosphorized  type, 
occur  in  the  urine  under  certain  conditions  (see  page  396).  In  this 
connection  we  will  use  the  term  nucleoprotein.  The  pathological  con- 
ditions under  which  the  content  of  nucleoprotein  is  increased  includes 
all  affections  of  the  urinary  passages  and  in  particular  pyelitis,  nephritis, 
and  inflammation  of  the  bladder. 

Experiments 

I.  Detection  of  Nucleoprotein. — Place  10  c.c.  of  urine  in  a  small  beaker, 
dilute  it  with  three  volumes  of  water  to  prevent  precipitation  of  urates,  and  make 
the  reaction  very  strongly  acid  with  acetic  acid.  If  the  urine  becomes  turbid 
it  is  an  indication  that  nucleoprotein  is  present. 

^  If  it  is  considered  desirable  to  test  for  peptone  the  proteose  may  be  removed  by  satu- 
ration with  (NH4)2SO<  according  to  the  directions  given  on  p.  120  and  the  filtrate  tested 
for  peptone  by  the  biuret  test. 


URINE  429 

If  the  urine  under  examination  contains  albumin  the  greater  portion  of  this 
substance  should  be  removed  by  boiling  the  urine  before  testing  it  for  the  pres- 
ence of  nucleoprotein. 

2.  Tannic  Acid  Precipitation  Test  (Ott). — Mix  25  c.c.  of  the  urine  with 
an  equal  volimie  of  a  saturated  solution  of  sodixmi  chloride  and  slowly  add 
Almen's  reagent.'  In  the  presence  of  nucleoprotein  a  voluminous  precipitate 
forms. 

BLOOD 

The  pathological  conditions  in  which  blood  occurs  in  the  urine  may 
be  classified  under  the  two  divisions  hematuria  and  honoglohimiria. 
In  hematuria  we  are  able  to  detect  not  only  the  hemoglobin  but  the 
unruptured  corpuscles  as  well,  whereas  in  hemoglobinuria  the  pig- 
ment alone  is  present.  Hematuria  is  brought  about  through  blood 
passing  into  the  urine  because  of  some  lesion  of  the  kidney  or  of  the 
urinary  tract  below  the  kidney.  Hemoglobinuria  is  brought  about 
through  hemolysis,  i.e.,  the  rupturing  of  the  stroma  of  the  erythrocyte 
and  the  liberation  of  the  hemoglobin.  This  may  occur  in  scurvy, 
t3rphus,  pyemia,  purpura,  and  in  other  diseases.  It  may  also  occur  as 
the  result  of  a  burn  covering  a  considerable  area  of  the  body,  or  may 
be  brought  about  through  the  action  of  certain  poisons  or  by  the  in- 
jection of  various  substances  having  the  power  of  dissolving  the 
erythrocytes.     Transfusion  of  blood  may  also  cause  hemoglobinuria. 

Even  in  true  hematuria  the  erythryocytes  may  escape  detection  if 
the  urine  is  ammoniacal  inasmuch  as  the  cells  disintegrate  under  these 
conditions. 

Experiments 

1.  Potassixim  Hydroxide  Test  (Heller) .—Render  loc.c.  of  urine  strongly  alka- 
line with  potassium  hydroxide  solution  and  heat  to  boiling.  Upon  allowing  the 
heated  urine  to  stand  a  precipitate  of  phosphates,  colored  red  by  the  contained 
hematin,  is  formed.  It  is  ordinarily  well  to  make  a  *'  control "  experiment  using 
normal  urine,  before  coming  to  a  final  decision. 

Certain  substances,  such  as  cascara  sagrada,  rhubarb,  santonin,  and  senna, 
cause  the  urine  to  give  a  similar  reaction.  Reactions  due  to  such  substances 
may  be  differentiated  from  the  true  blood  reaction  by  the  fact  that  both  the  pre- 
cipitate and  the  pigment  of  the  former  reaction  disappear  when  treated  with 
acetic  acid,  whereas  if  the  color  is  due  to  hematin  the  acid  will  only  dissolve  the 
precipitate  of  phosphates  and  leave  the  pigment  undissolved. 

2.  Teichmann's  Hemin  Test. — Place  a  small  drop  of  the  suspected  urine  or  a 
small  amount  of  the  moist  sediment  on  a  microscopic  sUde,  add  a  minute  grain 
of  sodimn  chloride  and  carefully  evaporate  to  dryness  over  a  low  flame.  Put  a 
cover-glass  in  place,  run  underneath  it  a  drop  of  glacial  acetic  acid,  and  warm 
gently  until  the  formation  of  gas  bubbles  is  observed.  Cool  the  preparation, 
examine  under  the  microscope,  and  compare  the  form  of  the  crystals  with  those 

1  Dissolve  5  grams  of  tannic  acid  in  240  c.c.  of  50  per  cent  alcohol  and  add  10  c.c.  of  25 
per  cent  acetic  acid. 


43©  PHYSIOLOGICAL   CHEMISTRY 

reproduced  in  Figs.  78  and  79,  page  265.     (See  Atkinson  and  Kendall's  and 
Nippe's  modifications,  page  264.) 

3.  Heller-Teichmann  Reaction. — Produce  the  pigmented  precipitate  according 
to  directions  given  in  Heller's  test  above.  If  there  is  a  copious  precipitate  of  phos- 
phates and  but  little  pigment  the  phosphates  may  be  dissolved  by  treatment  with 
acetic  acid  and  the  residue  used  in  the  formation  of  the  hemin  crystals  according 
to  directions  in  Experiment  2,  above. 

4.  V.  Zeynek  and  Nencki's  Hemin  Test. — To  10  c.c.  of  the  urine  under  examina- 
tion add  acetone  until  no  more  precipitate  forms.  Filter  ofif  the  precipitate  and 
extract  it  with  10  c.c.  of  acetone  rendered  acid  with  2-3  drops  of  hydrochloric  acid. 
Place  a  drop  of  the  resulting  colored  extract  on  a  slide,  immediately  place  a  cover- 
glass  in  position,  and  examine  under  the  microscope.  Compare  the  form  of  the 
crystals  with  those  shown  in  Figs.  78  and  79,  page  265.  Hemin  crystals  produced 
by  this  manipulation  are  sometimes  very  minute,  thus  rendering  it  difficult  to  de- 
termine the  exact  form  of  the  crystal. 

6.  Guaiac  Test. — Place  5  c.c.  of  urine  in  a  test-tube  and  by  means  of  a 
pipette  introduce  a  freshly  prepared  alcoholic  solution  of  guaiac  (strength  about 
I  :  60)  into  the  fluid  until  a  turbidity  results,  then  add  old  turpentine  or  hydro- 
gen peroxide,  drop  by  drop,  until  a  blue  color  is  obtained. 

This  is  a  very  delicate  test  when  properly  performed.  Buckmaster 
has  suggested  the  use  of  guaiaconic  acid  instead  of  the  solution  of 
guaiac.  The  test  is  positive  both  before  and  after  boiling  the  blood 
for  15-20  seconds.  Pus  does  not  respond  after  boiling.  Old,  partly 
putrefied  pus  gives  the  test  even  without  the  addition  of  hydrogen 
peroxide  or  old  turpentine  whereas /res/?  pus  responds  upon  the  addition 
of  hydrogen  peroxide.     See  discussion  on  page  258  and  test  on  page  262. 

7.  Schxunm's  Modification  of  the  Guaiac  Test. — To  about  5  c.c.  of  lurine^  in  a 
test-tube  add  about  10  drops  of  a  freshly  prepared  alcoholic  solution  of  guaiac. 
Agitate  the  tube  gently,  add  about  20  drops  of  old  turpentine,  subject  the  tube  to 
a  thorough  shaking,  and  permit  it  to  stand  for  about  2-3  minutes.  A  blue  color 
indicates  the  presence  of  blood  in  the  solution  under  examination.  In  case  there 
is  not  sufficient  blood  to  yield  a  blue  color  under  these  conditions,  a  few  cubic  centi- 
meters of  alcohol  should  be  added  and  the  tube  gently  shaken,  whereupon  a  blue 
coloration  will  appear  in  the  upper  alcohol-turpentine  layer. 

A  control  test  should  always  be  made  using  water  in  place  of  urine.  In  the 
detection  of  very  minute  traces  of  blood  only  3-5  drops  of  the  guaiac  solution  should 
be  employed.  '\ 

8.  Ortho-Tolidin  Test  (Ruttan  and  Hardisty).^ — To  i  c.c.  of  a  4  per  cent 
glacial  acetic  acid  solution  of  o-tolidin'  in  a  test-tube  add  i  c.c.  of  the  solution 

^  Alkaline  urine  should  be  made  sHghtly  acid  with  acetic  acid  as  the  blue  end-reaction 
is  very  sensitive  to  alkaU. 

2  Ruttan  and  Hardisty:  Canadian  Medical  Assn.  Journal,  Nov.,  191 2,  also  Biochemical 
Bull.,  2,  22s,  1913. 

"NHz  NH2 

\  / 

CsH^  —  C6H4 

/  \ 

CHj  CH3 


URINE  431 

under  examination  and  i  c.c.  of  3  per  cent  hydrogen  peroxide.  In  the  presence 
of  blood  a  bluish  color  develops  (sometimes  rather  slowly)  and  persists  for  some- 
time (several  hours  in  some  instances). 

This  test  is  said  to  be  as  sensitive  for  the  detection  of  occult  blood 
in  feces  and  stomach  contents  as  is  the  benzidine  reaction.  It  is  also 
claimed  to  be  more  satisfactory  for  urine  than  any  other  blood  test. 
The  acetic  acid  solution  may  be  kept  for  one  month  with  no  reduction 
in  delicacy. 

9.  Benzidine  Reaction. — This  is  one  of  the  most  delicate  of  the  reac- 
tions for  the  detection  of  blood.  Different  benzidine  preparations  vary 
greatly  in  their  sensitiveness,  however.  Inasmuch  as  benzidine  solu- 
tions change  readily  upon  contact  with  light,  it  is  essential  that  they 
be  kept  in  a  dark  place. 

The  test  is  peformed  as  follows:  To  a  saturated  solution  of  benzidine  in 
alcohol  or  glacial  acetic  acid  add  an  equal  volume  of  3  per  cent  hydrogen  peroxide 
and  I  c.c.  of  the  urine  under  examination.  If  the  mixture  is  not  already  acid, 
render  it  so  with  acetic  acid,  and  note  the  appearance  of  a  blue  color.  A 
control  test  should  be  made  substituting  water  for  the  urine. 

Often  when  urines  containing  a  small  amount  of  blood  are  tested  by 
this  reaction,  the  mixture  is  rendered  so  turbid  as  to  make  it  difl&cult  to 
decide  as  to  the  presence  of  a  faint  green  color.  Such  urines  should 
be  washed  with  water  before  the  test  is  applied  to  it.  The  sensitiveness 
of  the  benzidine  reaction  is  greater  when  applied  to  aqueous  solutions 
than  when  applied  to  the  urine. 

For  a  modification  of  this  test  and  further  discussion  see  Chapter 
XV  on  Blood  and  Lymph. 

9.  Spectroscopic  Examination. — Submit  the  urine  to  a  spectroscopic  exami- 
nation according  to  the  directions  given  on  page  296,  looking  especially  for  the 
absorption  bands  of  oxyhemoglobin  and  methemoglobin  (see  Absorption  Spectra, 
Plate  I). 

PUS 

Pus  may  be  present  in  the  urine  in  inflammatory  affections  of 
various  types.  Such  a  condition  is  termed  pyuria.  Albumin  always 
accompanies  the  pus.  In  catarrh  of  the  bladder  and  in  inflammation 
of  the  urethra  or  of  the  pelvis  of  the  kidney  pus  is  particularly  apt  to 
be  present  in  the  urine.  If  a  urine  of  high  pus  concentration  is  voided 
it  may  indicate  the  rupturing  of  an  abscess  in  some  part  of  the  genito- 
urinary tract.  Pus  may  be  detected  by  one  of  the  procedures  given 
below. 

Experiments 

I .  Microscopical  Detection  of  Pus. — The  characteristic  form  elements  of  pus  are 
leucocytes.     They  may  occur  in  very  small  number  in  normal  urine.     Examine  the 


432  PHYSIOLOGICAL   CHEMISTRY 

urine  (centrifugated  if  necessary)  under  the  microscope.  Any  considerable  number 
of  pus  corpuscles  indicates  a  pathological  urine.  In  acid  urine  the  pus  corpuscles 
appear  as  round,  colorless  cells,  composed  of  refractive,  granular  protoplasm. 
Sometimes  they  may  exhibit  amoeboid  movements,  particularly  if  the  slide  contain- 
ing them  be  warmed  slightly.  They  are  nucleated  (one  or  more  nuclei),  the  nuclei 
being  clearly  visible  only  upon  treating  the  cells  with  water,  acetic  acid  or  some 
other  suitable  reagent.  In  alkaline  urine  the  pus  corpuscles  are  often  degenerated. 
They  may  occur  as  swollen,  transparent  cells,  which  exhibit  no  granular  structure. 
If  the  degeneration  has  proceeded  far  enough  the  nuclei  fade  and  the  cell  disinte- 
grates and  only  debris  remains. 

Sometimes  it  is  almost  impossible  to  differentiate  between  pus  corpuscles  and 
certain  types  of  epithelial  cells.  In  such  a  case  apply  one  of  the  following  chemical 
tests. 

2.  Guaiac  Test. — This  test  is  not  specific  for  pus,  but  is  given  by  certain 
other  substances  and  particularly  by  blood  (see  Chapter  XV).  Perform  the  test 
as  follows :  Acidify  the  urine  (if  alkaline)  with  acetic  acid,  filter, ^  and  add  tinc- 
ture of  guaiac  to  the  sediment  on  the  paper.  If  the  pus  is  old,  and  partly  putrefied 
it  will  give  a  blue  color.  If  no  blue  color  is  secured,  add  old  turpentine,  or  hy- 
drogen peroxide,  drop  by  drop.  A  blue  color  formed  only  under  these  conditions 
indicates  fresh  pus. 

As  a  control  test  boil  some  of  the  urine  (or  sediment)  for  15-20  seconds  and 
repeat  the  test.  Pus  does  not  respond  after  boiling.  In  the  case  of  blood  the 
test  is  positive  both  before  and  after  boiUng. 

3.  Potassium  Hydroxide  Test  (Donne). — Separate  the  sediment  from  the 
urine  (by  decantation,  filtration  or  centrifugation) ;  place  a  small  piece  of  solid 
potassium  hydroxide  on  the  sediment  and  stir.  If  pus  is  present  (and  particu- 
larly if  it  be  fresh  pus  and  not  disintegrated)  the  sediment  will  become  slimy  and 
tough.  If  the  sediment  is  mucus  it  will  more  or  less  pass  into  solution  in  the 
concentrated  alkali. 

BILE 

Both  the  pigments  and  the  acids  of  the  bile  may  be  detected  in  the 
urine  under  certain  pathological  conditions.  Of  the  pigments,  bilirubin 
is  the  only  one  which  has  been  positively  identified  in  fresh  urine;  the 
other  pigments,  when  present,  are  probably  derived  from  the  bilirubin. 
A  urine  containing  bile  may  be  yellowish-green  to  brown  in  color  and 
when  shaken  foams  readily.  The  staining  of  the  various  tissues  of  the 
body  through  the  absorption  of  bile  due  to  occlusion  of  the  bile  duct 
cause  a  condition  known  as  icterus  or  jaundice.  Bile  is  always  present 
in  the  urine  under  such  conditions  unless  the  amount  of  bile  reaching 
the  tissues  is  extremely  small. 

Experiments 

Tests  for  Bile  Pigments 

Practically  all  of  these  tests  for  bile  pigments  are  based  on  the 
oxidation  of  the  pigment  by  a  variety  of  reagents  with  the  formation 
1  If  desired,  the  urine  may  be  centrifuged  and  the  sediment  used  in  the  test. 


URINE  433 

of  a  series  of  colored  derivatives,  e.g.,  biliverdin  (green),   bilicyanin 
(blue),  choletelin  (yellow). 

1.  Gmelin's  Test— To  about  5  c.c.  of  concentrated  nitric  acid  in  a  test-tube 
add  an  equal  volume  of  urine  carefully  so  that  the  two  fluids  do  not  mix.  At  the 
point  of  contact  note  the  various  colored  rings,  green,  blue,  violet,  red,  and  red- 
dish-yellow. 

2.  Rosenbach's  Modification  of  Gmelin's  Test. — Filter  5  c.c.  of  urine  through 
a  small  filter  paper.  Introduce  a  drop  of  concentrated  nitric  acid  into  the  cone 
of  the  paper  and  observe  the  succession  of  colors  as  given  in  Gmelin's  test. 

3.  Nakayama's  Reaction. — To  5  c.c.  of  urine  in  a  test-tube  add  an  equal  volume 
of  a  10  per  cent  solution  of  barium  chloride.  Centrifugate  the  mixture,  pour  off 
the  supernatant  fluid,  and  heat  the  precipitate  with  2  c.c.  of  Nakayama's  reagent.^ 
In  the  presence  of  bUe  pigments  the  solution  assumes  a  blue  or  green  color. 

3.  Huppert's  Reaction. — Thoroughly  shake  equal  volumes  of  urine  and  milk  of 
lime  in  a  test-tube.  The  pigments  imite  with  the  calcium  and  are  precipitated. 
Filter  off  the  precipitate,  wash  it  with  water,  and  transfer  to  a  small  beaker.  Add 
alcohol  acidified  slightly  with  hydrochloric  acid  and  warm  upon  a  water-bath  untU 
the  solution  becomes  colored  an  emerald  green. 

According  to  Steensma,  this  procedure  may  give  negative  results  even  in  the 
presence  of  the  pigments,  owing  to  the  fact  that  the  acid-alcohol  is  not  a  suflBciently 
strong  oxidizing  agent.  He  therefore  suggests  the  addition  of  a  drop  of  a  0.5  per 
cent  solution  of  sodium  nitrite  to  the  acid-alcohol  mixture  before  warming  on  the 
water-bath.     Try  this  modification  also. 

4.  Salkowski's  Test — Render  5  c.c.  of  urine  alkaline  with  a  few  drops  of  a  10 
per  cent  sodium  carbonate  solution  and  add  a  10  per  cent  solution  of  calcium 
chloride,  drop  by  drop,  until  the  supernatant  fluid  exhibits  the  normal  urinary  color 
when  the  contents  of  the  test-tube  are  thoroughly  mixed.  Filter  oft  the  precipitate, 
and  after  washing  it  place  it  in  a  second  tube  with  95  per  cent  alcohol.  Acidify  the 
alcohol  with  hydrochloric  acid  and,  if  necessary,  shake  the  tube  to  bring  the  pre- 
cipitate into  solution.  Heat  the  solution  to  boiling  and  observe  the  appearance  of 
a  green  color  which  changes  through  blue  and  violet  to  red;  if  no  bile  is  present 
the  solution  does  not  undergo  any  color  change.  This  test  will  frequently  exhibit 
greater  delicacy  than  Gmelin's  test.  Steensma's  suggestions  mentioned  under 
Huppert's  Reaction,  above,  apply  in  connection  with  this  test  also. 

5.  Hammarsten's  Reaction. — To  about  5  c.c.  of  Hammarsten's  reagent-  in  a 
small  evaporating  dish  add  a  few  drops  of  urine.  A  green  color  is  produced.  If 
more  of  the  reagent  is  now  added  the  play  of  colors  as  noted  in  Gmelin's  test  may 
be  obtained. 

6.  Smith's  Test. — To  2-3  c.c.  of  urine  in  a  test-tube  add  carefully  about  5  c.c. 
of  dilute  tincture  of  iodine  (i  :  10)  so  that  the  fluids  do  not  mix.  A  green  ring  is 
observed  at  the  point  of  contact. 

7.  Salkowski-Schippers  Reaction. — XeutraUze  the  acidity  of  10  c.c.  of  the 
urine  under  examination  with  a  few  drops  of  a  dilute  solution  of  sodium  carbonate, 
and  add  5  drops  of  a  20  per  cent  solution  of  sodium  carbonate  and  10  drops  of  a  20 

'  Prepared  by  combining  99  c.c.  of  alcohol  and  i  c.c.  of  fuming  hydrochloric  acid  con- 
taining 4  grams  of  ferric  chloride  per  liter. 

^  Hammarsten'.s  reagent  is  made  by  mixing  i  volume  of  25  per  cent  nitric  acid  and  19 
volumes  of  25  per  cent  hydrochloric  acid  and  then  adding  i  volume  of  this  acid  mixture 
to  4  volumes  of  95  per  cent  alcohol. 
28 


434  PHYSIOLOGICAL   CHEMISTRY 

per  cent  solution  of  calcium  chloride.  Filter  off  the  resultant  precipitate  upon  a 
hardened  filter  paper  and  wash  it  with  water.  Remove  the  precipitate  to  a  small 
porcelain  dish,  add  3  c.c.  of  an  acid-alcohol  mixture^  and  a  few  drops  of  a  dilute 
solution  of  sodium  nitrite  and  heat.  The  production  of  a  green  color  indicates  the 
presence  of  bile  pigments. 

8.  Bonanno's  Reaction.  ^ — Place  5-10  c.c.  of  the  urine  under  examination  in  a 
smaU  porcelain  evaporating  dish  and  add  a  few  drops  of  Bonanno's  reagent.'  If 
bile  is  present  an  emerald-green  color  will  develop.  Bonanno  says  the  reaction  is 
not  interfered  with  by  any  known  normal  or  pathological  urinary  constituent. 

Tests  for  Bile  Acids 

1,  Sucrose — HjS04  Test  (Pettenkofer). — To  5  c.c.  of  urine  in  a  test-tube  add 
5  drops  of  a  5  per  cent  solution  of  sucrose.  Now  incline  the  tube,  run  about 
2-3  c.c.  of  concentrated  sulphmric  acid  carefully  down  the  side  and  note  the 
red  ring  at  the  point  of  contact.  Upon  slightly  agitating  the  contents  of  the 
tube  the  whole  soultion  gradually  assumes  a  reddish  color.  As  the  tube  be- 
comes warm,  it  should  be  cooled  in  running  water  in  order  that  the  tempera- 
ture may  not  rise  above  7o°C. 

It  is  claimed  that  this  test  is  not  satisfactory  in  the  presence  of 
protein  and  chromogenic  substances  which  yield  interfering  colors 
with  sulphuric  acid. 

2.  Furfural — H2SO4  Test  (Mylius). — To  approximately  5  c.c.  of  urine  in  a 
test-tube  add  3  drops  of  a  very  dilute  (i  :  1000)  aqueous  solution  of  furfural, 

HC CH 

HC        C.CHO. 


O 

Now  incline  the  tube,  run  about  2-3  c.c.  of  concentrated  sulphuric  acid  carefully 
down  the  side  and  note  the  red  ring  as  above.  In  this  case  also,  upon  shaking 
the  tube,  the  whole  solution  is  colored  red.  Keep  the  temperature  below  7o°C. 
as  before. 

3.  Foam  Test  (v.  Udransky). — To  5  c.c.  of  urine  in  a  test-tube  add  3-4  drops 
of  a  very  dilute  (i  :  1000)  aqueous  solution  of  furfural.  Place  the  thiunb  over  the 
top  of  the  tube  and  shake  until  a  thick  foam  is  formed.  By  means  of  a  small 
pipette  add  2-3  drops  of  concentrated  sulphuric  acid  to  the  foam  and  observe  the 
dark  pink  coloration  produced. 

4.  Surface  Tension  Test  (Hay). — This  test  is  based  upon  the  principle  that 
bile  acids  have  the  property  of  reducing  the  surface  tension  of  fluids  in  which 
they  are  contained.  The  test  is  performed  as  follows:  Cool  about  10  c.c.  of 
urine  in  a  test-tube  to  i7°C.  or  lower,  and  sprinkle  a  little  finely  pulverized  sul- 
ph\ir  upon  the  surface  of  the  fluid.     The  presence  of  bile  acids  is  indicated  if  the 

^  ^lade  by  adding  5  c.c.  of  concentrated  hydrochloric  acid  to  95  c.c.  of  96  per  cent, 
alcohol. 

^11  Tommasi,  2,  Xo.  21. 

'This  reagent  may  be  prepared  by  dissolving  2  grams  of  sodium  nitrite  in  100  c.c.  of 
concentrated  hydrochloric  acid. 


URINE  435 

sulphur  sinks  to  the  bottom  of  the  liquid,  the  rapidity  with  which  the  sulphur  sinks 
depending  upon  the  amount  of  bile  acids  present  in  the  urine.  The  test  is  said  to 
react  with  bile  acids  when  the  latter  are  present  in  the  proportion  i  :  120,000. 
Allen^  has  recently  suggested  the  quantitative  determination  of  bile  acids  by  a 
surface  tension  method. 

5.  Neukomm's  Modification  of  Pettenkofer's  Test. — To  a  few  drops  of  urine 
in  an  evaporating  dish  add  a  trace  of  a  dilute  sucrose  solution  and  one  or  more  drops 
of  dilute  sulphuric  acid.  Evaporate  on  a  water-bath  and  observe  the  development 
of  a  violet  color  at  the  edge  of  the  evaporating  mixture.  Discontinue  the  evapora- 
tion as  soon  as  the  color  is  observed. 

6.  Peptone  Test  (Oliver). — To  5  c.c.  of  urine  add  2-3  drops  of  acetic  acid, 
filtering  if  necessary.  Add  an  equal  volume  of  a  i  per  cent,  solution  of  Witte's 
peptone  to  the  acid  solut'on.  A  precipitate  is  formed  which  is  insoluble  in  an  excess 
of  acetic  acid.     This  precipitate  is  a  compound  of  protein  and  bile  acids. 

CH3 

! 

ACETONE,      C=0. 

CH3 

It  was  formerly  very  generally  believed  that  acetone  appeared  in  the 
urine  under  pathological  conditions  because  of  increased  protein  de- 
composition. It  is  now  generally  thought  that,  in  man,  the  output 
of  acetone  arises  principally  from  the  breaking  down  of  fatty  tissues 
or  fatty  foods  within  the  organism.  The  quantity  of  acetone  elimi- 
nated has  been  shown  to  increase  when  the  subject  is  fed  an  abundance 
of  fat-containing  food  as  well  as  during  fasting,  whereas  a  replace- 
ment of  the  fat  w^th  carbohydrates  is  followed  by  a  marked  decrease 
in  the  acetone  excretion.  If  no  carbohydrate  food  is  fed  the  output  of 
acetone  bodies  increases  at  once,  producing  a  physiological  acidosis 
(see  Chapter  XXVII  on  Metabolism). 

Acetone  and  the  closely  related  bodies,  i3-hydrox\'butyric  acid  and 
acetoacetic  acid,  are  generally  classified  as  the  acetone  bodies.  They 
are  all  associated  with  a  deranged  metabolic  function  and  may  appear 
in  the  urine  together  or  separately,  depending  upon  the  conditions. 
Acetone  and  diacetic  acid  may  occur  alone  in  the  urine  but  ^-hy- 
droxybutyric  acid  is  never  found  except  in  conjunction  with  one  or  the 
other  of  these  bodies.  Acetone  and  acetoacetic  acid  arise  chiefly 
from  the  oxidation  of  (3-hydroxybutyric  acid.  The  relation  existing 
between  these  three  bodies  is  shown  in  the  following  equations. 

(a)  CH3.CH(OH).CH2.COOH+O^CH3CO.CH2.COOH+H20. 

fl-hydroxybutyric  acid.  Acetoacetic  acid. 

(b)  CH3CO.CH2.COOH-^(CH3)2CO+COo. 

Acetoacetic  acid.  Acetone. 

^  Allen:  Jour.  Biol.  Cliem.,  22,  505,  1915. 


436  PHYSIOLOGICAL   CHEMISTRY 

Acetone,  chemically  considered,  is  a  ketone,  di-methyl  ketone.  When 
pure  it  is  a  liquid  which  possesses  a  characteristic  aromatic  fruit-like 
odor,  boils  at  56-5 7°C.  and  is  miscible  with  water,  alcohol,  or  ether 
in  all  proportions.  Acetone  is  a  physiological  as  well  as  a  pathological 
constituent  of  the  urine  and  under  normal  conditions  the  daily  output 
(preformed  acetone  +  acetoacetic  acid)  is  about  3-15  mg. 

Pathologically,  the  elimination  of  acetone  is  often  greatly  increased 
and  at  such  times  a  condition  of  acetonuria  is  said  to  exist.  Values 
from  0.02-6  grams  or  higher  have  been  obtained  for  preformed  acetone 
plus  acetone  derived  from  acetoacetic  acid.  This  pathological  ace- 
tonuria may  accompany  diabetes  mellitus,  scarlet  fever,  typhoid 
fever,  pneumonia,  nephritis,  phosphorus  poisoning,  grave  anemias, 
fasting,  and  a  deranged  digestive  function;  it  also  frequently  accom- 
panies auto-intoxication  and  chloroform  and  ether  anethesia.  The 
types  of  acetonuria  most  frequently  met  with  are  those  noted  in  febrile 
conditions  and  in  advanced  cases  of  diabetes  mellitus.  The  blood  in 
diabetic  comas  has  been  found  to  contain  as  high  as  45  mg.  of  total 
acetone  (acetone  +  acetoacetic  acid)  for  100  c.c.  of  blood  serum. 

Experiments 

1,  Isolation  from  the  Urine. — In  order  to  facilitate  the  detection  of  acetone  in 
the  urine,  the  specimen  under  examination  should  be  distilled  and  the  tests  as  given 
below  applied  to  the  resulting  distillate.  If  it  is  not  convenient  to  distil  the  urine, 
the  tests  may  be  conducted  upon  the  undistilled  fluid.  To  obtain  an  acetone  dis- 
tillate proceed  as  follows:  Place  100-250  c.c.  of  urine  in  a  distillation  flask  or  retort 
and  render  it  acid  with  acetic  acid.  Collect  about  one-third  of  the  original  volume 
of  fluid  as  a  distillate,  add  5  drops  of  10  per  cent  hydrochloric  acid  and  redistil  about 
one-half  of  this  volume.     With  this  final  distillate  conduct  the  tests  as  given  below. 

2.  Gunning's  Iodoform  Test. — To  about  5  c.c.  of  the  urine  or  distillate  in 
a  test-tube  add  a  few  drops  of  Lugol's  solution^  or  ordinary  iodine  solution  (I  in 
KI)  and  a  few  drops  of  dilute  NH4OH  to  form  a  black  precipitate  (nitrogen  iodide). 
Allow  the  tube  to  stand  (the  length  of  time  depending  upon  the  content  of 
acetone  in  the  fluid  under  examination)  and  note  the  formation  of  a  yellowish  sedi- 
ment consisting  of  iodoform.  Examine  the  sediment  under  the  microscope  and 
compare  the  form  of  the  crystals  with  those  shown  in  Fig.  8,  page  42. 

If  the  crystals  are  not  well  formed  recrystallize  them  from  ether 
and  examine  again.  The  crystals  of  iodoform  should  not  be  confounded 
with  those  of  calcium  phosphate  (Fig.  105,  page  322)  which  may  be  formed 
in  this  test,  particularly  if  made  upon  the  undistilled  urine.  This  test 
is  preferable  to  Lieben's  test  (4)  since  no  substance  other  than  acetone 
will  produce  iodoform  when  treated  according  to  the  directions  for 

^  Lugol's  solution  may  be  prepared  by  dissolving  4  grams  of  iodine  and  6  grams  of  potas- 
sium iodide  in  100  c.c.  of  distilled  water. 


URIXE  437 

this  test;  both  alcohol  and  aldehyde  yield  iodoform  when  tested  by 
Lieben's  test. 

Gunning's  test  is  rather  satisfactory  for  the  detection  of  acet<mc, 
and  has  been  used  with  good  results  even  upon  the  undistilled  urine. 
Protein  material  apparently  interferes  with  the  reaction,  and  when 
present  the  urine  should  be  distilled  and  the  distillate  used.^  In 
some  instances  where  the  amount  of  acetone  present  is  very  small  it 
is  necessary  to  allow  the  tube  to  stand  24  hours  before  making  the  ex- 
amination for  iodoform  crystals.  This  test  serves  to  detect  acetone 
when  present  in  the  ratio  i  :  100.000. 

3.  Sodium  Nitroprusside  Test  ('Legal;. — Introduce  about  5  c.c.  of  the  urine  or 
distillate  into  a  test-tube,  add  a  few  drops  of  freshly  prepared  aqueous  solution 
of  sodium  nitroprusside  and  render  the  mixture  alkaline  with  potassium  hydrox- 
ide. (Be  sure  to  add  the  nitroprusside  before  the  solution  is  rendered  alkaline.) 
A  ruby-red  color,  due  to  creatinine,  a  normal  urinary  constituent,  is  produced  (see 
Weyl's  test,  page  385J.  Add  an  excess  of  acetic  acid  and  if  acetone  is  present 
the  red  color  will  be  intensified,  whereas  in  the  absence  of  acetone  a  yellow  color 
will  result.     Make  a  control  test  upon  normal  urine  to  show  that  this  is  so. 

A  similar  red  color  may  be  produced  by  paracresol  in  urines  con- 
taining no  acetone. 

Two  hypotheses  have  been  proposed  to  explain  the  color  reaction 
between  acetone  and  nitroprusside:  (i)  The  formation  of  a  complex  ion 
of  ferropentacyanide  with  the  isonitroso  compound  of  the  ketone, 
or  (2)  the  formation  of  such  an  ion  with  the  isordtroamine  derivative 
of  the  ketone.  2 

4.  Iodoform  Test  (Lieben). — Introduce  5  c.c.  of  the  urine  or  distillate  into  a 
test-tube,  render  it  alkaUne  with  potassiimi  hydroxide  and  add  1-2  c.c.  of  iodine 
solution  drop  by  drop.  If  acetone  is  present  a  yellowish  precipitate  of  iodoform 
will  be  produced.  Identify  the  iodoform  by  means  of  its  characteristic  odor  and 
its  typical  crystalline  form  (see  Fig.  8,  page  42). 

While  fully  as  delicate  as  Gunning's  test  (2)  this  test  is  not  as 
accurate  since,  by  means  of  the  procedure  involved,  either  alcohol  or 
aldehyde  will  yield  a  precipitate  of  iodoform.  This  test  is  especially 
liable  to  lead  to  erroneous  deductions  when  urines  from  the  advanced 
stages  of  diabetes  are  under  examination,  because  of  the  presence  of 
alcohol  formed  from  the  sugar  through  fermentative  processes.'  If 
protein  is  present  in  the  urine  to  be  tested  it  may  prevent  the  acetone 
from  responding  to  the  above  reaction.     It  is  therefore  advisable  to  use 

'  Rosenbloom:  Jour.  Am.  Med.  Ass'n,  59,  445,  191 2. 

*Cambi:  Atli.  accad,  Lincei,  22,  376,  1913. 

'  Welker  reports  the  production  of  a  pink  or  red  color  during  the  application  of  this  test 
to  the  distillates  from  pathological  urines  which  had  been  preserved  with  powdered  thymol. 
He  found  the  color  to  be  due  to  an  iodothymol  compound  which  had  been  previously  pre- 
pared synthetically  by  Messinger  and  Vortmann. 


438  PHYSIOLOGICAL    CHEMISTRY 

the  distillate  to  secure  most  accurate  results.^     SobeP  has  suggested  a 
quantitative  method  for  acetone  based  on  Lieben's  test. 

5.  Reynolds -Gunning  Test. — This  test  depends  upon  the  solubility  of  mercuric 
oxide  in  acetone  and  is  performed  as  follows :  To  5  c.c.  of  the  urine  or  distillate  add 
a  few  drops  of  mercuric  chloride,  render  the  solution  alkaline  with  potassium  hy- 
droxide and  add  an  equal  volume  of  95  per  cent  alcohol.  Shake  thoroughly  in 
order  to  bring  the  major  portion  of  the  mercuric  oxide  into  solution  and  filter. 
Render  the  clear  filtrate  faintly  acid  with  hydrochloric  acid  and  stratify  some  am- 
monium sulphide  (NH4)2S  upon  this  acid  solution.  At  the  zone  of  contact  a 
blackish-gray  ring  of  precipitated  mercuric  sulphide,  HgS,  will  form.  Aldehyde 
also  responds  to  this  test.  Aldehyde,  however,  has  never  been  detected  in  the  urine 
and  could  be  present  in  this  instance  only  if  the  acidified  urine  was  distilled  too  far. 

6.  Rothera's  Reaction.^ — To  5-10  c.c.  of  urine  or  distillate  in  a  test-tube  add  a 
little  solid  ammonium  sulphate,  2-3  drops  of  a  freshly  prepared  5  per  cent  solution 
of  sodium  nitroprusside  and  1-2  c.c.  of  concentrated  ammonium  hydroxide.  The 
development  of  a  permanganate  color  indicates  the  presence  of  acetone. 

Hunter*  claims  that  this  reaction  serves  to  detect  acetoacetic  acid  rather  than 
acetone.     Others  claim  it  detects  both  acetone  and  acetoacetic  acid. 

8.  Salicylaldehyde  Reaction  (Frommer). — Render  10  c.c.  of  urine  strongly 
alkaline  with  potassium  hydroxide,  add  10-12  drops  of  a  10  per  cent  solution  of 
salicylaldehyde  in  absolute  alcohol  and  warm  the  mixture  to  about  70°.  K 
acetone  be  present  the  fluid  becomes  yellow,  then  red,  reddish-piurple  and  dark 
red  in  turn.  The  color  of  the  urine  is  practically  unchanged  if  no  acetone  is 
present. 

This  color  is  due  to  the  formation  of  dihydroxydibenzoylacetone 
through  the  interaction  of  saUcylaldehyde  and  acetone. 

CH3 

I 
ACETOACETIC  ACID,  C  =  0 

I 
CH2COOH. 

Acetoacetic  or  diacetic  acid  occurs  in  traces  in  normal  urine. 
The  sum  of  the  acetone  and  the  acetoacetic  acid  excreted  in  normal 
urine  per  day  ranges  from  3  to  15  mg.  and  ordinarily  three-quarters 
of  this  is  acetoacetic  acid.  Under  certain  pathological  conditions 
it  occurs  in  larger  quantities  and  is  rarely  found  except  associated 
with  acetone.  It  is  formed  from  jS-hydroxybutyric  acid,  another  of 
the  acetone  bodies,  and  upon  decomposition  yields  acetone  and  car- 
bon dioxide.  Acetoaceturia  occurs  ordinarily  under  the  same  condi- 
tions as  the  pathological  acetonuria,  i.e.,  in  fevers,  diabetes,  etc.  (pp. 
435  and  534).  If  very  little  acetoacetic  acid  is  formed  it  may  be 
transformed  into  acetone,  whereas  if  a  larger  quantity  is  produced  both 

^Rosenbloom:  Jour.  Am.  Med.  Ass'n,  59,  445,  1912. 
^Sobel:  Schweiz.  Apoth.  Zlg.,  52,  62,  1914. 
•Rothera:  Jour.  Physiol.,  37,  491,  1908. 
*  Hunter:  Quart.  Jour.  Exp.  Physiol.,  8,  13,  1914. 


URINE  439 

acetone  and  acetoacetic  acid  may  be  present  in  the  urine.  Aceto- 
aceturia  is  most  frequently  observed  in  children,  especially  accompany- 
ing fevers  and  digestive  disorders;  it  is  perhaps  less  frequently  observed 
in  adults,  but  when  present,  particularly  in  fevers  and  diabetes  it  is 
frequently  followed  by  fatal  coma. 

Acetoacetic  acid  is  a  colorless  liquid  which  is  miscible  with  water, 
alcohol  and  ether,  in  all  proportions.  It  differs  from  acetone  in  giving 
a  violet-red  or  Bordeaux-red  color  with  a  dilute  solution  of  ferric 
chloride. 

Experiments^ 

1.  Le  Nobel  Reaction.- — Make  lo  c.c.  of  urine  add  with  acetic  acid,  add  a 
few  drops  of  a  dilute  aqueous  solution  of  sodium  nitroprusside  and  stratify  con- 
centrated ammonium  hydroxide  upon  the  mixture.  In  the  presence  of  aceto- 
acetic acid  a  violet  ring  forms  at  once. 

Acetone  also  responds  to  this  test,  but  the  test  is  more  delicate  for  aceto- 
acetic acid  and  the  response  is  more  prompt. 

2.  Ferric  Chloride  Test  (Gerhardt). — To  5  c.c.  of  urine  in  a  test-tube  add 
ferric  chloride  solution,  drop  by  drop,  until  no  more  precipitate  forms.  In  the 
presence  of  acetoacetic  acid  a  Bordeatix-red  color  is  produced;  this  color  may  be 
somewhat  masked  by  the  precipitate  of  ferric  phosphate,  in  which  case  the  fluid 
should  be  filtered. 

A  positive  result  from  the  above  manipulation  simply  indicates  the  possible 
presence  of  acetoacetic  acid.  Before  making  a  final  decision  regarding  the  pres- 
ence of  this  body  make  the  two  following  control  experiments : 

(aj  Place  5  c.c.  of  urine  in  a  test-tube,  small  beaker,  or  Erlenmeyer  flask 
and  boil  it  vigorously  for  3-5  minutes.  Cool  the  vessel  and,  with  the  boiled 
urine,  make  the  test  as  given  above.  As  has  been  already  stated,  acetoacetic 
acid  yields  acetone  upon  decomposition  and  acetone  does  not  give  a  Bordeaux- 
red  color  with  ferric  chloride.  By  boiling  as  indicated  above,  therefore,  any 
acetoacetic  acid  present  would  be  decomposed  into  acetone  and  carbon  dioxide 
and  the  test  upon  the  resulting  fluid  would  be  negative.  If  positive,  the  color 
is  due  to  the  presence  of  bodies  other  than  acetoacetic  acid. 

(b)  Place  5  c.c.  of  urine  in  a  test-tube,  acidify  with  HjS04,  to  free  aceto- 
acetic acid  from  its  salts,  and  carefully  extract  the  mixture  with  ether  by  shaking. 
If  acetoacetic  acid  is  present  it  will  be  extracted  by  the  ether.  Now  remove 
the  ethereal  solution,  evaporate  it  to  dryness,  dissolve  the  residue  in  1-2  c.c. 
of  water  and  add  3-5  drops  of  3  per  cent  ferric  chloride.  Acetoacetic  acid  is 
indicated  by  the  production  of  the  characteristic  Bordeaux-red  color. 

This  color  disappears  spontaneously  in  24-48  hours.  Such  sub- 
stances as  antipyrin,  kairin,  phenacetin,  saHcyhc  acid,  saHcylates, 
sodium  acetate,   thiocyanates,  and  thallin  yield  a  similar  red   color 

^  To  prepare  a  diacetic  acid  solution  which  may  be  added  to  urine,  if  urines  containing 
this  acid  are  not  available,  proceed  as  follows:  Treat  13  grams  of  ethyl  acetoacetate  with 
500  c.c.  of  N/5  sodium  hydroxide.  Allow  to  stand  for  48  hours  to  hydrolyze'the  ester. 
In  preparing  urine  for  tests  add  i  part  of  this  solution  to  10  parts  of  urine. 

^Harding  and  Ruttan:  Biochem.  Jour.,  6,  445,  1912;  also  Biochem.  Bull.,  2,  223,  1913. 


440  PHYSIOLOGICAL   CHEMISTRY 

under  these  conditions,  but  when  due  to  the  presence  of  any  of  these 
substances  the  color  does  not  disappear  spontaneously  but  may  re- 
main permanent  for  days.  Many  of  these  disturbing  substances  are 
soluble  in  benzene  or  chloroform  and  may  be  removed  from  the  urine 
by  this  means  before  extracting  with  ether  as  above.  Acetoacetic 
acid  is  insoluble  in  benzene  or  chloroform. 

3.  Sodium  Nitrite — ^Ferrous  Sulphate  Reaction  (Hurtley). — Place  10  c.c. 
of  urine  in  a  large  test-tube,  add  21/2  c.c.  of  concentrated  hydrochloric  acid 
and  I  c.c.  of  fresh  i  per  cent  sodium  nitrite.  Shake  the  tube  and  permit  it  to 
stand  for  two  minutes.  Add  15  c.c.  of  concentrated  ammonium  hydroxide  and 
5  c.c.  of  10  per  cent  ferrous  sulphate.  Shake  the  tube  and  permit  it  to  stand. 
Note  the  slow  development  of  a  violet  or  purple  color  in  the  presence  of  aceto- 
acetic acid. 

This  test  serves  to  detect  acetoacetic  acid  when  present  in  a  dilu- 
tion of  I  to  50,000.  The  concentration  of  the  acetoacetic  acid  regulates 
the  speed  at  which  the  color  develops.  If  the  concentration  be  very 
low  an  interval  of  five  hours  may  elapse  before  the  color  appears. 
The  test  is  believed  to  be  specific  for  acetoacetic  acid. 

4.  Arnold -Lipliawsky  Reaction. — This  reaction  is  somewhat  more  delicate  than 
Gerhardt's  test  (2)  and  serves  to  detect  acetoacetic  acid  when  present  in  the  pro- 
portion of  I  125,000.  It  is  also  negative  toward  acetone,  /?-hydroxybutyric  acid 
and  the  interfering  drugs  mentioned  as  causing  erroneous  deductions  in  the  applica- 
tion of  Gerhardt's  test.  If  the  urine  under  examination  is  highly  pigmented  it 
should  be  partly  decolorized  by  means  of  animal  charcoal  before  applying  the  test 
as  indicated  below. 

Place  5  c.c.  of  the  urine  under  examination  and  an  equal  volume  of  the  Arnold- 
Lipliawsky  reagent^  in  a  test-tube,  add  a  few  drops  of  concentrated  ammonia  and 
shake  the  tube  vigorously.  Note  the  production  of  a  brick-red  color.  '  Take  1-2 
c.c.  of  this  colored  solution,  add  10-20  c.c.  of  hydrochloric  acid  (sp.  gr.  1.19),  3  c.c. 
of  chloroform,  and  2-4  drops  of  ferric  chloride  solution  and  carefully  mix  the  fluids. 
Acetoacetic  acid  is  indicated  by  the  chloroform  assuming  a  violet  or  blue  color;  if 
acetoacetic  acid  is  absent  the  color  may  be  yellow  or  light  red. 

H    OHH 
^-HYDROXYBUTYRIC  ACID,  H — C — C — C — COOH. 

H    H    H 

This  acid  occurs  in  normal  urine  in  traces,  e.g.,  20-30  mg.  jper  day.^ 

It  is  found  under  certain  pathological  conditions  in  larger  quantities 

^  This  reagent  consists  of  two  definite  solutions  which  are  ordinarily  preserved  separately 
and  mixed  just  before  using.     The  two  solutions  are  prepared  as  follows: 

(a)  One  per  cent  aqueous  solution  of  potassium  nitrite. 

(b)  One  gram  of  ^-amino-acetophenon  dissolved  in  100  c.c.  of  distilled  water  and 
enough  hydrochloric  acid  (about  ?  c.c.)  added,  drop  by  drop,  to  cause  the  solution,  which 
is  at  first  yellow,  to  become  entirely  colorless.     An  excess  of  acid  must  be  avoided. 

Before  using,  a  and  b  are  mixed  in  the  ratio  1:2. 

2  Shaffer  and  Marriott:  Jour.  Biol.  C/iem.,  16,  265,  1913. 


URINE  44 1 

and  then  always  in  conjunction  with  either  acetone  or  acetoacetic  acid. 
Either  of  these  bodies  may  be  formed  from  /3-hydroxybutyric  acid  under 
proper  conditions.  It  is  present  in  especially  large  amount  in  severe 
cases  of  diabetes  and  has  also  been  detected  in  digestive  disturbances, 
continued  fevers,  scurvy,  measles,  and  in  fasting.  It  is  probable 
that,  in  man,  /S-hydroxybut^Tic  acid,  in  common  with  acetone  and 
acetoacetic  acid,  arises  principally  from  the  breaking  down  of  fatty 
tissues  mthin  the  organism.  Any  condition  in  which  large  amounts 
of  acetone  and  acetoacetic  acid,  and  in  severe  cases  /3-hydroxybutyric 
acid  also,  are  excreted  in  the  urine  is  known  as  an  "acidosis."  In 
diabetes  the  deranged  metabolic  conditions  cause  the  production  of 
great  quantities  of  these  suDstances  which  lead  to  an  acid  intoxication 
and  ultimately  to  diabetic  coma.  In  severe  diabetes  50-100  grams  or 
over  per  day  may  be  excreted.  In  such  conditions  the  jS-hydroxy- 
butyric  acid  may  constitute  60-80  per  cent  of  the  total  acetone  bodies. 
In  rare  cases  we  may  have  an  excretion  of  large  amounts  of  j8-hydroxy- 
butyric  acid  with  a  low  acetone  output.  An  acidosis  may  also  occur 
under  certain  physiological  conditions  (see  Chapter  XXVII  on  Metab- 
olism). 

Ordinarily  jS-hydroxybutyric  acid  is  an  odorless,  transparent  syrup 
which  is  levorotatory  and  easily  soluble  in  water,  alcohol,  and  ether; 
it  may  be  obtained  in  crystalline  form. 

Experiments 

I.  Black's  Reaction.' — Inasmuch  as  the  urinary  pigments  as  well  as  any 
contained  sugar  or  acetoacetic  acid  will  interfere  with  the  delicacy  of  this  test 
when  applied  to  the  urine  directly,  the  following  preliminary  procedure  is  neces- 
sary:  Concentrate  10  c.c.  of  the  urine  under  examination  to  one-third  or  one- 
fourth  of  its  original  volume  in  an  evaporating  dish  at  a  gentle  heat.  Acidify 
the  residue  with  a  few  drops  of  concentrated  hydrochloric  acid,  add  sufficient 
plaster  of  Paris  to  make  a  thick  paste  and  allow  the  mixture  to  stand  until  it 
begins  to  "set."  It  should  now  be  stirred  and  broken  up  in  the  dish  by  means 
of  a  stirring  rod  with  a  blunt  end.  Extract  the  porous  meal  thus  produced  twice 
with  ether  by  stirring  and  decantation.  Any  ,3-hydroxybutyric  acid  present 
will  be  extracted  by  the  ether.  Evaporate  the  ether  extract  spontaneously  or 
on  a  water-bath,  dissolve  the  residue  in  water,  and  neutralize  it  with  barium 
carbonate.  To  5  to  10  c.c.  of  this  neutral  fluid  in  a  test-tube  add  2  to  3 
drops  of  ordinary  commercial  acid  hydrogen  peroxide.  Mix  by  shaking  and 
add  a  few  drops  of  Black's  reagent.-  Permit  the  tube  to  stand  and  note  the 
gradual  development  of  a  rose  color  which  increases  to  its  maximum  intensity 
and  then  gradually  fades.  ^ 

^  Black:  Jour.  Biol.  C/tem.,  5,  207,  1908. 

*  Made  by  dissolving  5  grams  of  ferric  chloride  and  0.4  gram  of  ferrous  chloride  in  100 
c.c.  of  water. 

^  This  disappearance  of  color  is  due  to  the  further  o.xidation  of  the  acetcacedc  acid. 


44 2  PHYSIOLOGICAL   CHEMISTRY 

In  carrying  out  the  test  care  should  be  taken  to  see  that  the  solution 
is  cold  and  approximately  neutral  and  that  a  large  excess  of  hydrogen 
peroxide  and  Black's  reagent  are  not  added.  In  case  but  little 
/3-hydroxybutyric  acid  is  present  the  color  will  fail  to  appear  or  will 
be  but  transitory  if  the  oxidizing  agents  are  added  in  too  great  excess. 
It  is  preferable  to  add  a  few  drops  of  the  reagent  and  at  intervals  of 
a  few  minutes  repeat  the  process  until  the  color  undergoes  no  further 
increase  in  intensity.  One  part  of  /3-hydroxybutyric  acid  in  10,000 
parts  of  the  solution  may  be  detected  by  this  test. 

2.  Polariscopic  Examination. — Subject  some  of  the  urine  (free  from  protein) 
to  the  ordinary  fermentation  test  (see  page  421).  This  will  remove  glucose 
and  fructose,  which  would  interfere  with  the  polariscopic  test.  Now  examine  the 
fermented  fluid  in  the  polariscope  and  if  it  is  levorotatory  the  presence  of  /3-hy- 
droxybutyric acid  is  indicated.  This  test  is  not  absolutely  reliable,  however,  since 
conjugate  glycuronates  are  also  levorotatorj^  after  fermentation. 

CONJUGATE  GLYCURONATES 

Glycuronic  acid  does  not  occur  free  in  the  urine,  but  is  found,  for 
the  most  part,  in  combination  with  phenol.  Much  smaller  quantities 
are  excreted  in  combination  with  indoxyl  and  skatoxyl.  The  total 
content  of  conjugate  glycuronates  seldom  exceeds  0.004  per  cent  Under 
normal  conditions.  The  output  may  be  very  greatly  increased  as 
the  result  of  the  administration  of  antipyrin,  borneol,  camphor, 
chloral  hydrate,  menthol,  morphine,  naphthol,  turpentine,  etc.  The 
glycuronates  as  a  group  are  levorotatory  whereas  glycuronic  acid  is 
dextrorotatory.  Most  of  the  glycuronates  reduce  alkaUne  metallic 
oxides  and  so  introduce  an  error  in  the  examination  of  urine  for  sugar. 
Conjugate  glycuronates  often  occur  associated  with  glucose  in  glyco- 
suria, diabetes  mellitus,  and  in  some  other  disorders.  As  a  class  the 
glycuronates  are  non-fermentable. 

Experiments 

1.  Naphthoresorcinol  Reaction  (ToUens). — Introduce  5  c.c.  of  urine  in  a 
test-tube  and  add  0.5-1  c.c.  of  a  i  per  cent  solution  of  naphthoresorcinol  in 
95  per  cent  alcohol,  and  5  c.c.  of  concentrated  hydrochloric  acid.  Raise  the 
temperature  gradually  to  the  boiling-point  and  boil  for  one  minute,  shaking  the 
tube  continuously.  Stand  the  tube  aside  four  minutes,  then  cool  vmder  the 
tap.  Extract  with  an  equal  volume  of  ether.  Glycuronates  are  indicated  by 
the  ether  extract  assuming  a  violet-red  color.  The  spectroscope  shows  this 
extract  to  possess  two  absorption  bands,  one  on  the  D  line  and  one  to  the  right 
of  this  line. 

2.  Polariscopic-Fermentation  Test. — If  glucose  is  present  in  the  urine  tested 


URINE  443 

for  glycuronates  the  urine  may  first  be  subjected  to  a  polariscopic  examination, 
then  fermented  and  a  second  polariscopic  examination  made.  The  sugar  being 
dextrorotatory  and  fermentable  and  the  glycuronates  being  levorotatory  and 
non-fermentable  the  second  polariscopic  test  will  show  a  levorotation  indicative 
of  conjugate  glycuronates. 

3.  Reduction-Polariscopic  Test. — Test  the  urine  by  FehUng's  test.  If 
positive  try  the  Resorcinol-HCl  reaction  for  fructose.  If  negative  test  the  optical 
activity.     Levorotation  indicates  glycuronates. 

PENTOSES 

We  have  two  distinct  types  of  pentosuria,  i.e.,  alimentary  pentosuria, 
resulting  from  the  ingestion  of  large  quantities  of  pentose-rich  vegetables 
such  as  prunes,  cherries,  grapes,  or  plums,  and  fruit  juices,  in  which 
condition  the  pentoses  appear  only  temporarily  in  the  urine;  and  the 
chronic  form  of  pentosuria,  in  which  the  output  of  pentoses  bears  no 
relation  whatever  to  the  quantity  and  nature  of  the  pentose  content 
of  the  food  eaten.  In  occurring  in  these  two  forms,  pentosuria  re- 
sembles glycosuria  (see  page  413),  but  it  is  definitely  known  that  pen- 
tosuria bears  no  relation  to  diabetes  melhtus  and  there  is  no  generally 
accepted  theory  to  account  for  the  occurrence  of  the  chronic  form  of 
pentosuria.  The  pentose  detected  most  frequently  in  the  urine  is 
arabinose,  the  inactive  form  generally  occurring  in  chronic  pentosuria 
although  keto-pentose^  may  occur  in  some  cases.  The  levorotatory 
variety  occurs  in  the  aHmentary  t^-pe  of  the  disorder. 

Experiments 

1.  Orcinol-Hydrochloric  Acid  Reaction  (Bial).- — To  5  c.c.  of  Bial's  reagent' 
in  a  test-tube  add  2-3  c.c.  of  urine  and  heat  the  mixture  gently  until  the  first 
bubbles  rise  to  the  surface.^  Immediately  or  upon  cooling  the  solution  becomes 
green  and  a  fiocculent  precipitate  of  the  same  color  may  form. 

This  test  is  believed  to  be  more  accurate  than  the  original  orcinol 
test.  It  is  claimed  that  urines  containing  menthoL  kreosotal,  etc., 
respond  to  the  old  orcinol  reaction,  but  not  to  Bial's.  If  so  desired 
the  osazone  of  the  pentose  may  be  formed,  then  distilled  with  hydro- 
chloric acid  and  the  distillate  tested  by  Bial's  test  (Jolles). 

2.  Phloroglucinol-Hydrochloric  Acid  Reaction  (Tollensi. — To  equal  volxmies 
of  urine  and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  Uttle  phloroglucinol  and  heat 
the  mixture  on  a  boiling  water-bath.    Pentose,  galactose,  or  glycuronic  acid 

'Levene  and  La  Forge:  Jour.  Biol.  Cliem.,  18,  319,  1Q14. 

'Bial:  Deut.  tned.  Woch.,  28,  252,  1902 

'Orcinol ...         15  grams. 

Fuming  HCl 500  grams. 

Ferric  chloride  (10  per  cent) 20-30  drops. 

*  The  test  may  also  be  performed  by  adding  the  urine  to  the  hot  reagent.  No  further 
heating  should  be  necessary  if  pentose  is  present. 


444  PHYSIOLOGICAL    CHEMISTRY 

will  be  indicated  by  the  appearance  of  a  red  color.  To  differentiate  between 
these  bodies  examine  by  the  spectroscope  and  look  for  the  absorption  band 
between  D  and  E  given  by  pentoses  and  glycuronic  acid,  and  then  differentiate 
between  the  two  latter  bodies  by  the  melting-points  of  their  osazones. 

3.  Orcinol  Test. — Place  equal  volumes  of  urine  and  hydrochloric  acid  (sp.  gr. 
1.09)  in  a  test-tube,  add  a  smaU  amount  of  orcinol,  and  heat  the  mixture  to  boiling. 
Color  changes  from  red  through  reddish-blue  to  green  will  be  noted.  When  the 
solution  becomes  green  it  should  be  shaken  in  a  separatory  funnel  with  a  little 
amyl  alcohol,  and  the  alcoholic  extract  examined  spectroscopically.  An  absorption 
band  between  C  and  D  will  be  observed. 

FAT 

When  fat  finds  its  way  into  the  urine  through  a  lesion  which  brings 
some  portion  of  the  urinary  passages  into  communication  with  the 
lymphatic  system  a  condition  known  as  chyluria  is  established.  The 
turbid  or  milky  appearance  of  such  urine  is  due  to  its  content  of  chyle. 
This  disease  is  encountered  most  frequently  in  tropical  countries,  but 
is  not  entirely  unknown  in  more  temperate  climates.  Albumin  is  a 
constant  constituent  of  the  urine  in  chyluria.  Upon  shaking  a  chylous 
urine  with  ether  the  fat  is  dissolved  by  the  ether  and  the  urine  becomes 
clearer  or  entirely  clear. 

HEMATOPORPHYRIN 

Urine  containing  this  body  is  occasionally  met  with  in  various 
diseases,  but  more  frequently  after  the  use  of  quinine,  tetronal,  trional, 
and  especially  sulphonal.  Such  urines  ordinarily  possess  a  reddish 
tint,  the  depth  of  color  varying  greatly  under  different  conditions. 

Experiments 

1.  Spectroscopic  Examination. — ^To  100  c.c.  of  urine  add  about  20  c.c.  of 
a  10  per  cent  solution  of  potassium  hydroxide  or  ammonium  hydroxide.  The 
precipitate  which  forms  consists  principally  of  earthy  phosphates  to  which  the 
hematoporphyrin  adheres  and  is  carried  down.  Filter  off  the  precipitate,  wash 
it  and  transfer  to  a  flask  and  warm  with  alcohol  acidified  with  hydrochloric  acid. 
By  this  process  the  hematoporphyrin  is  dissolved  and  on  filtering  will  be  found 
in  the  filtrate  and  may  be  identified  by  means  of  the  spectroscope  (see  page 
296,  and  Absorption  Spectra,  Plate  11), 

2.  Acetic  Acid  Test. — To  100  c.c.  of  urine  add  5  c.c.  of  glacial  acetic  acid  and 
allow  the  mixture  to  stand  48  hours.  Hematoporphyrin  deposits  in  the  form  of  a 
precipitate. 

LACTOSE 

Lactose  is  rarely  found  in  the  urine  except  as  it  is  excreted  by  women 
during  pregnancy,  during  the  nursing  period,  or  soon  after  weaning. 


URINE  445 

It  is  rather  difficult  to  show  the  presence  of  lactose  in  the  urine  in  a 
satisfactory  manner,  since  the  formation  of  the  characteristic  lactos- 
azone  is  not  attended  with  any  great  measure  of  success  under  these 
conditions.  It  is,  however,  comparatively  easy  to  show  that  it  is  not 
glucose,  for,  while  it  responds  to  reduction  tests,  it  does  not  ferment 
with  pure  yeast  and  does  not  give  a  glucosazone.  An  absolutely 
conclusive  test,  of  course,  is  the  isolation  of  the  lactose  in  crystalline 
form  (Fig.  104,  page  318)  from  the  urine. 

On  oxidation  with  nitric  acid  lactose  and  galactose  yield  mucic  acid. 
This  test  is  frequently  used  in  urine  examination  to  differentiate  lactose 
and  galactose  from  other  reducing  sugars.  To  differentiate  lactose 
from  pentose,  since  neither  ferments,  we  may  apply  the  Orcinol— HCl 
test  of  Bial,  see  page  443. 

Experiments 

1.  Mucic  Acid  Test. — Treat  100  c.c.  of  the  urine  under  examination  with 
20  c.c.^  of  concentrated  nitric  acid  and  evaporate  the  mixture  in  a  broad,  shallow 
glass  vessel,  upon  a  boiling  water-bath  until  the  volimie  of  the  solution  is  only 
about  20  c.c.  At  this  point  the  fluid  should  be  clear  and  a  fine  white  precipitate 
of  mucic  acid  should  separate. 

If  the  percentage  of  lactose  in  the  urine  is  low  it  may  be  necessary 
to  cool  the  solution  and  permit  it  to  stand  for  some  time  before  the 
precipitate  \\-ill  form.  It  is  impossible  to  differentiate  between  galactose 
and  lactose  by  means  of  this  test,  but  the  reaction  does  serve  to  dif- 
ferentiate these  two  sugars  from  all  other  reducing  sugars.  A  sat- 
isfactory differentiation  between  lactose  and  galactose  in  pure  solution 
may  be  made  by  means  of  Barfoed's  test,  page  30.  This  test  is, 
however,  not  suited  for  urine  examination.  To  differentiate  galactose 
and  lactose  in  urine  use  the  Phloroglucinol-Hydrochloric  Acid  Reaction 
of  Tollens,  see  pages  36  and  443. 

2.  Rubner's  Test. — To  10  c.c.  of  urine  in  a  small  beaker  add  some  lead  acetate, 
in  substance,  heat  to  boiling,  and  add  NH4OH  until  no  more  precipitate  is  dissolved. 
In  the  presence  of  lactose  a  brick-red  or  rose-red  color  develops,  whereas  glucose 
gives  a  coffee-brown  color,  maltose  a  light  yellow  color,  and  fructose  no  color  at  all 
under  the  same  conditions. 

3.  Compotmd  Test. — Try  the  Nylander  reaction.  If  positive  tr>-  the  phenyl- 
hydrazine  test.  If  negative  (the  lactosazone  is  not  readily  formed  in  urine)  apply 
the  fermentation  test.  If  this  test  is  also  negative,  differentiate  between  lactose 
and  pentose  by  Orcinol-HCl  reaction  (Bial)  and  mucic  acid  tests. 

^  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35  c.c.  of 
nitric  acid.  Under  these  conditions  the  mixture  should  be  evaporated  until  the  remaining 
volume  is  appro.ximately  equivalent  to  that  of  the  nitric  acid  added. 


44^  PHYSIOLOGICAL   CHEMISTRY 

GALACTOSE 

Galactose  has  occasionally  been  detected  in  the  urine,  and  in  par- 
ticular in  that  of  nursing  infants  afiflicted  with  a  deranged  digestive 
function.  Lactose  and  galactose  may  be  differentiated  from  other 
reducing  sugars  which  may  be  present  in  the  urine  by  means  of  the 
mucic  acid  test.  This  test  simply  consists  in  the  production  of  mucic 
acid  through  oxidation  of  the  sugar  with  nitric  acid. 

V  Experiments 

1.  Mucic  Acid  Test. — Treat  loo  c.c.  of  the  urine  under  examination  with 
20  c.c.^  of  concentrated  nitric  acid  and  evaporate  the  mixture  in  a  broad,  shallow 
glass  vessel,  upon  a  boiling  water-bath,  until  the  volume  of  the  solution  is  only 
20  c.c.  At  this  point  the  fluid  should  be  clear  and  a  fine,  white  precipitate  of 
mucic  acid  should  separate. 

If  the  percentage  of  galactose  present  in  the  urine  is  low  it  may  be 
necessary  to  cool  the  solution  and  permit  it  to  stand  for  some  time 
before  the  precipitate  will  form.  It  is  impossible  to  differentiate 
between  galactose  and  lactose  by  means  of  this  test,  but  the  reaction 
does  serve  to  differentiate  these  two  sugars  from  all-  other  reducing 
sugars.  A  satisfactory  differentiation  between  galactose  and  lactose 
may  be  made  by  the  Phloroglucinol-Hydrochloric  Acid  Test  of  Tollens, 
below. 

2.  Phloroglucinol-Hydrochloric  Acid  Reaction  (Tollens). — To  equal  volumes  of 
the  urine  and  hydrochloric  acid  (sp.  gr.  1.09)  add  a  little  phloroglucinol  and  heat 
the  mixture  on  a  boiling  water-bath.  Galactose,  pentose,  and  glycuronic  acid  will 
be  indicated  by  the  appearance  of  a  red  color.  Galactose  may  be  differentiated 
from  the  two  latter  substances  in  that  its  solutions  exhibit  no  absorption  bands 
upon  spectroscopical  examination. 

FRUCTOSE 

Diabetic  urine  frequently  possesses  the  power  of  rotating  the  plane  of 
polarized  light  to  the  left,  thus  indicating  the  presence  of  a  levorotatory 
substance.  The  levorotation  is  sometimes  due  to  the  presence  of 
fructose,  although  not  necessarily  confined  to  this  carbohydrate,  since 
conjugate  glycuronates  and  j8-hydroxybutyric  acid,  two  other  levo- 
rotatory bodies,  are  frequently  found  in  the  urine  of  diabetics.  Fructose 
is  invariably  accompanied  by  glucose  in  diabetic  urine,  but  Jruc- 
tosuria  has  been  observed  as  a  separate  anomaly.     The  presence  of 

^  If  the  specific  gravity  of  the  urine  is  1020  or  over  it  is  necessary  to  use  25-35  c.c.  of 
nitric  acid.  Under  these  conditions  the  mixture  should  be  evaporated  until  the  remaining 
volume  is  approximately  equivalent  to  that  of  the  nitric  acid  added. 


URINE  447 

fructose  may  be  inferred  when  the  percentage  of  sugar,  as  determined 
by  the  titration  method,  is  greater  than  the  percentage  indicated  by 
the  polariscopic  examination. 

Experiments 

1.  Borchardt's  Reaction. — To  about  5  c.c.  of  urine  in  a  test-tube  add  an  equal 
volume  of  25  per  cent  hydrochloric  acid  and  a  few  crystals  of  resorcinol.  Heat  to 
boiling  and  after  the  production  of  a  red  color,  cool  the  tube  under  running  water 
and  transfer  to  an  evaporating  dish  or  beaker.  Make  the  mixture  sUghtly  alka- 
line with  solid  potassium  hydroxide,  return  it  to  a  test-tube,  add  2-3  c.c.  of 
acetic  ether,  and  shake  the  tube  vigorously.  In  the  presence  of  fructose  the 
acetic  ether  is  colored  yellow. 

The  only  urinary  constituents  which  interfere  with  the  test  are 
nitrites  and  indican  and  these  interfere  only  when  they  are  simul- 
taneously present.  Under  these  conditions,  the  urine  should  be  acidified 
with  acetic  acid  and  heated  to  boiling  for  one  minute  to  remove  the 
nitrites.  In  case  the  indican  content  is  very  large,  it  will  impart  a  blue 
color  to  the  acetic  ether,  thus  masking  the  yellow  color  due  to  fructose. 
When  such  urines  are  to  be  examined,  the  indican  should  first  be  re- 
moved by  Obermayer's  test  (see  page  388).  The  chloroform  should 
then  be  discarded,  the  acid-urine  mixture  diluted  with  one-third  its 
volume  of  water,  and  the  test  applied  as  described  above.  The  urine 
of  patients  who  have  ingested  santonin  or  rhubarb  responds  to  the  test. 
The  test  will  serve  to  detect  fructose  when  present  in  a  dilution  of 
I  :  2000,  i.e.,  0.05  per  cent. 

2.  Resorcinol-Hydrochloric  Acid  Reaction  (Seliwanofif). — To  5  c.c.  of  SeUwa- 
nofif's  reagent^  in  a  test-tube  add  a  few  drops  of  the  urine  under  examination 
and  heat  the  mixture  to  boiling.  The  presence  of  fructose  is  indicated  by  the 
production  of  a  red  color  and  the  separation  of  a  red  precipitate.  The  latter 
may  be  dissolved  in  alcohol  to  which  it  will  impart  a  striking  red  color. 

If  the  boiling  be  prolonged  a  similar  reaction  may  be  obtained  with 
urines  containing  glucose.  This  has  been  explained-  in  the  case  of 
glucose  as  due  to  the  transformation  of  the  glucose  into  fructose  by 
the  catalytic  action  of  the  hydrochloric  acid.  The  precautions  neces- 
sary for  a  positive  test  for  fructose  are  as  follows:  The  concentration  of 
the  hydrochloric  acid  must  not  be  more  than  12  per  cent.  The  reac- 
tion (red  color)  and  the  precipitate  must  be  observed  after  not  more  than 
20-30  seconds  of  boiling.  Glucose  must  not  be  present  in  amounts 
exceeding  2  per  cent.  The  precipitate  must  be  soluble  in  alcohol  with 
a  bright  red  color. 

'  Seliwanofif's  reagent  may  be  prepared  by  dissolving  0.05  gram  of  resorcinol  in  100  c.c. 
of  dilute  (i:  2)  hydrochloric  acid. 

^Koenigsfeld:  Bioch.  Zeit.  38,  311,  1912. 


448  PHYSIOLOGICAL   CHEMISTRY 

3.  Phenylhydrazine  Test. — Make  the  test  according  to  directions  under  Glu- 
cose, 3,  page  22. 

4.  Polariscopic  Examination. — A  simple  polariscopic  examination,  when  taken 
in  connection  with  other  ordinary  tests,  will  furnish  the  requisite  data  regarding 
the  presence  of  fructose,  provided  fructose  is  not  accompanied  by  other  levorotatory 
substances,  such  as  conjugate  glycuronates  and  )3-hydroxybutyric  acid. 

ARSENIC 

When  any  soluble  form  of  arsenic  is  introduced  into  the  body  in 
any  way,  it  is  quickly  absorbed  and  distributed  by  the  blood  and 
lymph.  The  absorption  is  influenced  by  the  quantity  and  quality  of 
the  food  in  the  stomach,  and  the  activity  of  the  circulation  of  the  part 
in  contact  with  the  poison.  Some  of  the  absorbed  arsenic  may  be 
returned  to  the  alimentary  canal  by  way  of  the  bile  and  gastro-intes- 
tinal  mucous  membrane.  After  absorption  it  may  be  deposited  in  the 
Uver,  kidneys,  brain,  bone,  muscles,  and  walls  of  the  stomach  and 
intestines.  It  is  eHminated  in  all  of  the  excretions,  but  chiefly  by  the 
kidneys  and  through  the  feces.  It  does  not  appear  very  promptly  in 
the  urine  but  continues  to  be  excreted  in  the  urine  over  a  long  period 
of  time,  in  some  cases  for  several  months.  The  urine  may  be  examined 
for  arsenic  by  the  following  methods. 

I.  Marsh  and  Marsh-Berzelius  Method. — This  method  has  the  advantage  of 
serving  as  a  qualitative  and  quantitative  determination,  and  is  a  very  delicate  test ; 
it  is,  however,  long  and  tedious.  The  various  steps  in  the  analysis  are:  (i)  the 
destruction  of  the  organic  matter  in  the  urine;  (2)  treatment  with  sulphuric  acid  to 
drive  off  excess  nitric  acid  and  break  up  nitro-compounds;  and  (3)  application  of 
independent  test  to  the  resultant  solution.  Proceed  as  follows:  The  urine,  to 
which  is  added  one-third  its  volume  of  nitric  acid,  is  placed  in  a  casserole  or  evapo- 
rating dish  and  evaporated  at  150°  to  160°  to  a  syrupy  consistency.  The  mass  is 
then  allowed  to  cool  and  5  c.c.  concentrated  sulphuric  acid  added,  and  gentle  heat 
applied.  The  heating  must  be  done  cautiously,  or  deflagration  takes  place  and 
some  of  the  arsenic  is  sure  to  be  lost.  The  mass  will  liquefy  and  finally  darken, 
indicating  organic  matter.  Cool  and  add  concentrated  nitric  acid,  i  c.c,  and  apply 
very  gentle  heat;  copious  reddish-brown  fumes  are  evolved.  Gradually  raise  the 
temperature  until  darkening  of  the  solution  occurs,  then  cool,  add  i  c.c.  concentrated 
nitric  acid  and  again  apply  gentle  heat,  and  repeat  the  process  until  the  solution 
faUs  to  darken.  Now  raise  the  temperature  until  white  fumes  begin  to  come  off. 
At  this  temperature  excess  nitric  acid  will  have  been  removed  and  all  nitro-com- 
pounds broken  up.  The  solution  at  this  point  is  clear  and  at  most  a  pale  straw  color. 
Cool  and  add  a  mixture  of  10  c.c.  concentrated  sulphuric  acid  and  40  c.c.  water, 
and  test  for  arsenic  using  a  Marsh  apparatus.  The  apparatus  (see  Fig.  131,  p.  449) 
consists  of  a  wide-mouth  flask — 250  c.c.  capacity — fitted  with  a  two-hole  stopper. 
Through  one  hole  is  passed  the  stem  of  a  separatory  funnel  of  50  to  60  c.c.  capacity. 
Through  the  other  hole  a  piece  of  glass  tube  bent  at  right  angles,  which  is  fitted  to 
a  calcium  chloride  tube,  and  this  in  turn  to  a  narrow  quartz  tube,  the  distal  end  of 


URINE 


449 


which  is  drawn  to  a  fine  bore  and  bent  up  almost  at  a  right  angle.  All  joints  must 
be  air-tight. 

Introduce  30  to  40  grams  of  arsenic-free  granulated  zinc  into  the  flask,  insert 
the  stopper  and  through  the  funnel  introduce  50  c.c.  dilute  sulphuric  acid  (i  part 
to  4  parts  water).  After  a  few  minutes  collect  a  test-tube  of  gas  by  inverting  a 
test-tube  over  the  end  of  the  quartz  tube,  and  test  it  by  igniting.  When  the  gas  in 
the  test-tube  ignites  quietly,  light  the  gas  issuing  from  the  quartz  tube. 

Hold  a  clean  porcelain  crucible  lid  in  the  flame  and  note  whether  any  deposit 
occurs.  This  precaution  must  be  taken  to  insure  that  the  chemicals  and  apparatus 
are  not  contaminated  with  arsenic. 

Now  introduce  the  prepared  urine  solution  into  the  funnel  and  adjust  the  flow 
so  that  6  to  8  drops  are  introduced  into  the  flask  per  minute.  Immediately  hold  a 
clean  porcelain  crucible  lid  in  the  flame  and  at  the  first  evidence  of  a  dark  deposit 
apply  heat,  using  a  wing-top  burner,  to  the  quartz  tube.  The  arsenic  if  present 
will  deposit  in  the  quartz  tube  beyond  the  flame.  Now  test  the  spot  on  the  lid 
to  see  if  it  is  arsenic;  it  should  dissolve  readily  in  sodium  hypochlorite  solution. 


.jZ. 


Fig.  131. — Marsh  Apparatus. 


Continue  the  operation  for  two  hours,  remove  the  Bunsen  burner  and  again  hold 
the  lid  in  the  flame.  If  no  more  deposits  on  the  lid,  the  arsenic  has  all  come  over 
and  is  deposited  in  the  quartz  tube;  if  deposition  occurs,  apply  the  Bunsen  again 
and  repeat. 

When  complete,  remove  the  quartz  tube,  weigh  it  after  cooling,  then  dissolve 
out  the  arsenic  with  nitric  acid,  wash,  dry,  and  weigh  again.  The  difference  in 
weight  is  the  weight  of  metallic  arsenic  in  the  volume  of  urine  taken. 

2.  Reinsch's  Test. — This  test  is  very  much  simpler,  but  not  so  delicate.  It 
has  the  advantage  of  application  in  the  presence  of  organic  matter.  The  test  is 
performed  as  follows:  The  urine,  acidified  with  one-fifth  its  volume  of  pure  hydro- 
chloric acid,  is  placed  in  a  beaker.  A  piece  of  bright  copper  foil  free  from  arsenic 
is  then  introduced,  and  the  urine  heated  almost  to  the  boiling-point.  It  is  then 
set  aside  for  six  to  eight  hours.  The  arsenic  is  deposited  on  the  copper  foil,  bluish- 
gray  color.  The  foil  is  then  removed,  washed  successively  in  pure  water,  alcohol, 
ether,  and  dried  without  heat.  The  foil  is  then  rolled  into  a  scroll  and  inserted 
into  a  3  mm.  bore  glass  tube  4  inches  long,  about  i  inch  from  the  end.  The  tube  is 
29 


450  PHYSIOLOGICAL   CHEMISTRY 

then  held  in  the  bunsen  flame  at  an  angle  of  20  to  25  degrees  applying  heat  where 
the  copper  foil  is  situated.  The  arsenic  volatilizes  and  is  oxidized,  and  deposits  as 
octahedral  crystals  of  arsenic  trioxide  on  the  cooler  part  of  the  tube.  The  crystals 
can  readily  be  recognized  by  the  microscope  and  sometimes  with  a  simple  magnify- 
ing lens. 

Mercury 

The  rapidity  of  absorption  of  mercury  depends  upon  a  number  of 
conditions  such  as,  mode  of  administration,  the  nature  of  the  com- 
pound and  its  physical  state,  the  state  and  condition  of  the  stomach 
and  intestines,  the  quantity  and  quaHty  of  the  food  in  the  stomach 
and  the  state  of  the  circulation  of  the  portal  of  entrance.  There  is 
no  definite  knowledge  as  to  the  form  in  which  it  is  absorbed.  Elimina- 
tion depends  upon  the  state  of  the  excretory  organs.  It  is  eliminated 
as  an  albuminate  in  all  the  excretions  of  the  body,  urine,  feces,  saliva, 
sweat,  tears,  and  milk.  Elimination  begins  about  two  hours  after 
introduction.  Depending  upon  the  amount  introduced  and  absorbed, 
the  time  required  for  its  complete  elimination  varies  from  24  hours  to 
many  weeks.  Mercury  may  be  detected  in  the  urine  by  the  following 
methods. 

1.  Reinsch's  Test. — The  procedure  is  carried  out  in  the  same  manner  as  for 
arsenic  (see  above).  A  piece  of  arsenic-free  copper  foil  is  introduced  into  the  urine 
acidified  with  one-fifth  its  volume  of  pure  hydrochloric  acid.  The  urine  is,  how- 
ever, not  heated  to  boUing,  but  warmed  to  50°  or  60°  and  set  aside  for  12  hours 
or  preferably  24  hours.  Metallic  mercury  is  deposited  on  the  foil  as  a  bright  lus- 
trous mirror.  The  foil  is  then  washed  with  pure  water,  alcohol,  ether,  and  dried 
without  heat,  rolled  into  a  scroll,  inserted  into  a  glass  tube  and  heated  in  the  same 
manner  as  under  arsenic.  The  mercury  is  deposited  in  the  metallic  state  in  the 
form  of  globules  readily  distinguished  with  the  microscope. 

2.  Amalgamation  Test. — A  more  rapid  method  than  the  above  is  by  amalga- 
mation with  zinc.  Add  5  grams  of  zinc  dust  to  the  urine  and  heat  for  15  minutes, 
stirring  continuously.  Allow  the  amalgamated  zinc  to  settle  and  decant  the  urine. 
Then  wash  by  decantation  several  times  with  pure  water,  then  with  alcohol,  and 
finally  with  ether  and  dry  in  air.  Now  introduce  the  dry  zinc  into  a  narrow  dry 
glass  tube  sealed  at  one  end.  With  the  Bunsen  soften  the  tube  about  2  inches 
above  the  zinc  and  constrict  the  tube  by  pulling  the  ends  apart.  Introduce  a  small 
bit  of  glass  wool  or  asbestos  sufficient  to  support  a  small  piece  of  iodine.  Intro- 
duce the  iodine  supported  by  the  asbestos  at  the  constriction.  Apply  heat  to  the 
zinc  amalgam,  and  then  gently  to  the  region  holding  the  iodine  to  gently  volatilize 
it,  and  immediately  reapply  heat  to  the  zinc.  The  mercury  volatilizes  and  meeting 
the  iodine  vapor  unites  with  it,  and  is  deposited  as  the  red  iodide  of  mercury. 

CHOH 


HOHC      CHOH 

INOSITOL,  I  I 

HOHC      CHOH 


CHOH 


URINE  451 

Inositol  occasionally  occurs  in  the  urine  in  albuminuria,  diabetes 
mellitus,  and  diabetes  insipidus.  It  is  claimed  also  that  copious  water- 
drinking  causes  this  substance  to  appear  in  the  urine.  Inositol  was  at 
one  time  considered  to  be  a  sugar  but  is  now  known  to  be  hexahy- 
droxybenzene,  as  the  above  formula  indicates.  It  is  an  example  of  a 
non-carbohydrate  in  whose  molecule  the  H  and  O  are  present  in  the 
proportion  to  form  water.  In  other  words  it  has  the  formula  of  the 
hexoses,  i.e.,  C6H12O6.  Inositol  occurs  widely  distributed  in  the 
vegetable  kingdom,  and  because  of  this  fact  the  theory  has  been  voiced 
that  it  represents  one  of  the  first  stages  in  the  conversion  of  a  car- 
bohydrate into  the  benzene  ring.  It  is  found  in  the  liver,  spleen, 
lungs,  brain,  kidneys,  suprarenal  capsules,  muscles,  leucocytes,  testes, 
and  urine  under  normal  conditions. 

Experiment 

I.  Detection  of  Inositol  (Scherer). — Acidify  the  urine  with  concentrated  nitric 
acid  and  evaporate  nearly  to  dryness.  Add  a  few  drops  of  ammonium  hydroxide 
and  a  little  calcium  chloride  solution  to  the  moist  residue  and  evaporate  the  mixture 
to  dryness.     In  the  presence  of  inositol  (o.ooi  gram)  a  bright  red  color  is  obtained. 

For  a  more  satisfactory  test,  which  is  also  more  time-consuming,  see  Salkowski's^ 
modification  of  Scherer's  test. 

LAIOSE 

This  substance  is  occasionally  found  in  the  urine  in  severe  cases  of 
diabetes  mellitus.  By  some  investigators  laiose  is  classed  with  the 
sugars.  It  resembles  fructose  in  that  it  has  the  property  of  reducing 
certain  metallic  oxides  and  is  levorotatory,  but  differs  from  fructose 
in  being  amorphous,  non-fermentable,  and  in  not  possessing  a  sweet 
taste. 

MELANINS 

These  pigments  never  occur  normally  in  the  urine,  but  are  present 
under  certain  pathological  conditions,  their  presence  being  especially 
associated  with  melanotic  tumors.  Ordinarily  the  freshly  passed  urine 
is  clear,  but  upon  exposure  to  the  air  the  color  deepens  and  may  at 
last  be  very  dark  brown  or  black  in  color.  The  pigment  is  probably 
present  in  the  form  of  a  chromogen  or  melanogcn  and  upon  coming 
into  contact  with  the  air  oxidation  occurs,  causing  the  transforma- 
tion of  the  melanogen  into  melanin  and  consequently  the  darkening 
of  the  urine. 

It  is  claimed  that  melanuria  is  proof  of  the  formation  of  a  visceral 
'  Salkowski:  Zeit.  physiol.  chctit.,  69,  478,  1910. 


452  PHYSIOLOGICAL   CHEMISTRY 

melanotic  growth.  In  many  instances,  wdthout  doubt,  urines  rich  in 
indican  have  been  wrongly  taken  as  diagnostic  proof  of  melanuria. 
The  pigment  melanin  is  sometimes  mistaken  for  indigo  and  melanogen 
for  indican.  It  is  comparatively  easy  to  differentiate  between  indigo 
and  melanin  through  the  solubiUty  of  the  former  in  chloroform. 

In  rare  cases  melanin  is  found  in  urinary  sediment  in  the  form  of  fine 
amorphous  granules. 

Experiments 

1.  Ferric  Chloride  Reaction  (von  Jaksch-Pollak). — ^Add  a  few  drops  of 
ferric  chloride  solution  to  lo  c.c.  of  urine  in  a  test-tube  and  note  the  formation  of 
a  gray  color.  Upon  the  further  addition  of  the  chloride  a  dark  precipitate  forms, 
consisting  of  phosphates  and  adhering  melanin.  An  excess  of  ferric  chloride 
causes  the  precipitate  to  dissolve. 

This  is  the  most  satisfactory  test  for  the  indentification  of  melanin  in  the 
urine. 

2.  Bromine  Test  (Zeller). — To  50  c.c.  of  urine  in  a  small  beaker  add  an  equal 
volume  of  bromine  water.  In  the  presence  of  melanin  a  yellow  precipitate  will 
form  and  will  gradually  darken  in  color,  ultimately  becoming  black. 

UROROSEIN 

This  is  a  pigment  which  is  not  present  in  normal  urine  but  may 
be  detected  in  the  urine  in  various  diseases,  such  as  pulmonary  tuber- 
culosis, typhoid  fever,  nephritis,  and  stomach  disorders.  Urorosein, 
in  common  with  various  other  pigments,  does  not  occur  preformed  in 
the  urine,  but  is  present  in  the  form  of  a  chromogen,  which  is  trans- 
formed into  the  pigment  upon  treatment  with  a  mineral  acid.  Herter^ 
showed  this  chromogen  to  be  indole  acetic  acid, 

H 
I 
iCCCOOH 

H 

Normal  urine  responds  to  the  urorosein  reaction  (see  below)  if  nitrites 
are  present. 

Experiments 

I.  Nitrite -Hydrochloric  Acid  Test  (Urorosein  Reaction). — To  10  c.c.  of  urine 
in  a  test-tube  add  2  c.c.  of  concentrated  hydrochloric  acid  and  a  few  drops  of  a 
I  per  cent  solution  of  potassiimi  nitrite.    A  rose-red  color  indicates  urorosein. 

The  chromogen  (indole  acetic  acid)  has  been  changed  into  urorosein 
by  oxidation. 

1  Herter:  Jour.  Biol.  Chem.,  4,  253,  1908. 


URINE  453 

2.  Robin's  Reaction. — Acidify  lo  c.c.  of  urine  with  about  15  drops  of  con- 
centrated hydrochloric  acid.  Upon  allowing  the  acidified  urine  to  stand,  a  rose-red 
color  will  appear  if  urorosein  is  present. 

3.  Nencki  and  Sieber's  Reaction. — To  100  c.c.  of  urine  in  a  beaker  add  10  c.c. 
of  25  per  cent  sulphuric  acid.  Allow  the  acidified  urine  to  stand  and  note  the  ap- 
pearance of  a  rose-red  color.  The  pigment  may  be  separated  by  extraction  with 
amyl  alcohol. 

NEPHROROSEIN 

This  pigment  is  closely  related  to  urorosein^  and  like  urorosein  it  is 
produced  from  a  chromogen  when  the  urine  is  treated  with  nitric  acid 
or  with  concentrated  hydrochloric  acid  and  a  Uttle  sodium  nitrite 
solution.  It  is  sometimes  called  /3-urorosein  to  differentiate  it  from  the 
true  urorosein  which  is  termed  a-urorosein.  Xephrorosein  occurs  only 
in  pathological  urines. 

UROCHROMOGEN 

This  is  the  chromogen  of  urochrome,  the  normal  urinary  pigment 
(see  Chapter  XXI).  It  is  claimed  that  the  iirochromogen  reaction  of  the 
urine  is  an  aid  to  prognosis  and  diagnosis  of  pulmonary  tuberculosis. 
Urochromogen  is  not  present  in  normal  urine.  Its  presence  in  patho- 
logical urine  is  due  probably  to  faulty  oxidation,  i.e.,  failure  to  oxi- 
dize the  chromogen  to  urochrome.  Urochromogen  ma}-  be  detected  by 
oxidizing  it  to  urochrome  by  means  of  potassium  permaganate.  In 
this  process  a  certain  antecedent  of  urochromogen  is  also  oxidized 
to  urochrome.  Whereas  the  diazo  reaction  (see  page  454)  is  also 
given  by  urines  containing  urochromogen,  it  is  claimed  that  the  diazo 
reaction  does  not  show  the  presence  of  the  precursor  of  urochromogen. 
Hence  the  urochromogen  reaction  is  said  to  be  more  constant  and  uni- 
form in  its  appearance. 

Experiment 

Urochromogen  Reaction  (Weisz).- — Fill  a  test-tube  a  little  less  than  one- 
third  full  of  urine,  dilute  it  with  2  volumes  of  distilled  water  and  mix  thoroughly. 
Pour  one-half  the  diluted  urine  into  another  tube  and  to  one  of  the  tubes  add  3 
drops  of  a  I  per  cent  solution  of  potassium  permanganate.  Shake  the  tube 
thoroughly.  In  the  presence  of  urochromogen  a  yellow  tint  will  appear  in  the 
tube  to  which  permanganate  was  added. 

The  reaction  is  due  to  the  oxidation  of  urochromogen  to  urochrome, 
and  is  believed  to  be  of  value  as  an  aid  in  prognosis  and  diagnosis  of 

'  Arnold:  Zeil.  physiol.  Chetn.,  71. 

-Weisz:  Munch,  med.  Woch.,  58,  1348,  igii. 

Vitri:  Seviana  Mcdica,  20,  No.  28,  1913. 

Hcflebower:  Am.  Jour.  Med.  Sci.,  143,  221,  1912. 

Metzger  and  Watson:  Jour.  Am.  Med.  Ass'n.,  62,  i8S6,  1914. 

Pignacca:  Gazeila  d.  Osp.  e  delle  Clin.,  25,  353,  191 4. 

Ferrannini:   Riforma  med.,  31,  479,  1915. 


454  PHYSIOLOGICAL    CHEMISTRY 

pulmonary  tuberculosis.  The  presence  of  sugar,  albumin  or  urobilin 
in  low  concentration  does  not  interfere  with  the  test.  The  test  often 
runs  parallel  with  the  diazo  reaction  (see  below.)  The  test  is  supposed 
to  be  positive  when  the  focus  of  the  lung  is  so  active  or  extensive  as  to 
flood  the  blood  with  toxins  or  to  break  down  the  defensive  forces  of  the 
body.  It  is  claimed,  therefore,  that  this  test  will  differentiate  the 
cases  in  which  the  tuberculosis  is  beyond  help  from  the  tuberculin 
from  those  in  which  the  body  is  liable  to  respond  favorably  to  its 
action.^  Some  investigators  claim  the  test  is  not  specific  and  that  a 
positive  reaction  will  be  obtained  in  many  disorders  other  than 
tuberculosis.^ 

UNKNOWN  SUBSTANCES 

I.  Ehrlich's  Diazo  Reaction. — Place  equal  volumes  of  urine  and  Ehrlich's 
diazobenzenesulphonic  acid  reagent^  in  a  test-tube,  mix  thoroughly  by  shaking, 
and  quickly  add  anmionium  hydroxide  in  excess.  The  test  is  positive  if  both  the 
fluid  and  the  foam  assume  a  red  color.  If  the  tube  is  allowed  to  stand  a  precipi- 
tate forms,  the  upper  portion  of  which  exhibits  a  blue,  green,  greenish-black,  or 
violet  color.  Normal  urine  gives  a  brownish-yellow  reaction  with  the  above 
manipulation. 

The  exact  nature  of  the  substance  or  substances  upon  whose  presence 
in  the  urine  this  reaction  depends  is  not  well  understood.  Some  in- 
vestigators claim  that  a  positive  reaction  indicates  an  abnormal  de- 
composition of  protein  material,  whereas  others  assume  it  to  be  due 
to  an  increased  excretion  of  alloxyproteic  acid,  oxyproteic  acid,  or  uro- 
ferric  acid.  Weisz^  claims  that  urochromogen  is  the  principal  urinary 
substance  which  causes  a  positive  diazo  reaction. 

The  reaction  may  be  taken  as  a  metabolic  symptom  of  certain  dis- 
orders, which  is  of  value  diagnostically  only  when  taken  in  connection 
with  the  other  symptoms.  The  reaction  appears  principally  in  the  urine 
in  febrile  disorders  and  in  particular  in  the  urine  in  typhoid  fever, 
tuberculosis,  and  measles.  The  reaction  has  also  been  obtained  in  the 
urine  in  various  other  disorders  such  as  carcinoma,  chronic  rheumatism, 

1  M.  and  A.  Weisz:  Wien.  klin.  Woch.,  25,  1183,  1912. 
Dozzi:  Gazetla  d.  Osp.  e  delle  Clin.,  34,  815,  1914. 
Burgess:  Jour.  Am.  Med.  Ass'n.,  66,  82,  1916. 

'^Tuliato:  Gazetta  d.  Osp.  e  delle  Clin.,  35,  1914. 
Martelli  and  Pizzetti:  Policlinico,  21,  April  i,  1914. 

'  Two  separate  solutions  should  be  prepared  and  mixed  in  definite  proportions  when 
needed  for  use: 

(a)  Five  grams  of  sodium  nitrite  dissolved  in  i  liter  of  distilled  water. 

ib)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid  in  i  liter  of  distilled 
water. 

Solutions  a  and  b  should  be  preserved  in  well-stoppered  vessels  and  mixed  in  the  propor- 
tion I  :  50  when  required,  Green  asserts  that  greater  delicacy  is  secured  by  mixing  the 
solutions  in  the  proportion  i  :ioo.  The  sodium  nitrite  deteriorates  upon  standing  and 
become?  unfit  for  ufc  in  the  course  of  a  few  weeks. 

*  Weisz:  Miinch.  mcd.  Woch.,  58,  1348,  1911. 


URINE  455 

diphtheria,    erysipelas,    pleurisy,    pneumonia,  scarlet  [fever,  syphilis, 
typhus,   etc.     The  administration  of   alcohol,   chrysarobin,   creosote, 
cresol,  dionin,  guaiacol,  heroin,  morphine,  naphthalene,  opium,  phenol, 
tannic  acid,  etc.,  will  also  cause  the  urine  to  give  a  positive  reaction. 
The  following  chemical  reactions  take  place  in  this  test: 

(a)  NaNOo+HCl^HXOo  +  XaCl. 

NH2  N 

/  /      % 

(b)  C6H4  +HNO>^C6H4  X+2H2O. 

\  \     / 

HSO3  SO3 

Sulphanilic  acid  Diazo-benzenesulphonic  acid. 

2.  MethyleneBlue  Reaction  (Russo).^ — To  5  c.c.  of  urine  add  4  drops  of  a  o.i  per 
cent  solution  of  methylene  blue.  In  cases  of  typhoid  fever,  measles,  smallpox  and 
certain  other  disorders  there  will  be  a  change  in  color  from  blue  to  green.  In 
normal  urine  the  blue  color  persists.  The  test  is  sometimes  used  as  a  substitute 
for  the  diazo  reaction  (see  p.  454). 

PHENOLSULPHONEPHTHALEIN  TEST  FOR  KIDNEY 

EFFICIENCY 

This  test  for  renal  function  was  devised  by  Rowntree  and  Geraghty.^ 
It  depends  upon  the  injection  into  the  tissues  of  a  dyestuff  which 
is^eliminated  rapidly  by  the  normal  kidneys,  and  can  be  easily  estimated 
quantitatively  in  the  urine. 

This  dyestuff,  phenolsulphonephthalein,  is  non-irritative  to  the 
body  either  when  taken  by  mouth  or  when  injected  into  the  tissues,^ 
so  that  it  does  no  harm  to  an  already  weakened  kidney. 

The  patient  upon  whom  the  test  is  to  be  performed  is  given  300-400 
c.c.  of  water  20-30  minutes  previously,  in  order  to  assure  a  free  flow 
of  urine. 

The  procedure  is  as  follows :  One  c.c.  of  a  solution  containing  6  mg.  of  phenol- 
sulphonephthalein^ is  injected  intramuscularly  in  the  lumbar  region,  the  time  of 
injection  being  noted.  The  patient  is  then  catheterized  and  the  urine  as  it  forms 
thereafter  allowed  to  drop  into  a  beaker  containing  2  drops  of  25  per  cent  NaOH. 
The  appearance  of  a  red  color  in  the  alkaUnized  urine  indicates  beginning  excre- 
tion of  the  drug,  the  normal  time  being  within  5  to  10  minutes  after  its  injection. 

1  Russo:  Kiforma  vied.,  Xo.  ig,  1905. 
Peskow:  Semainc  med.,  103,  1912. 
da  Pozzo:  Gaz.  Osp.  Clin.,  35,  865,  1914. 

^  Rowntree  and  Geraghty:  Jour.  Pliarm.  and  E.vper.  Therap.,  i,  579,  1910:  also  Arch. 
Int.  Med.,  March,  191 2,  p.  284. 

'Abel  and  Rowntree:  Jour.  Pharm.  and  E.xpcr.  Therap.,  i,  231,  1910. 

*  This  solution  is  prepared  by  adding  0.6  gram  phenolsulphonephthalein  and  0.84  c.c. 
of  2/N  NaOH  to  enough  0.75  per  cent  NaCl  solution  to  make  100  c.c.  This  gives  the  mono- 
sodium  or  acid  salt  which  is  slightly  irritant  locally  when  injected.  It  is  necessary  to  add 
2-3  drops  more  2/N  XaOH  which  changes  the  color  to  a  bordeau.x  red.  This_  prepara- 
tion  is  non-irritant. 


456  PHYSIOLOGICAL   CHEMISTRY 

Urine  is  now  collected  in  one-hour  samples.  In  patients  with  obstruction  to  the 
flow  of  urine  from  the  bladder  the  retention  catheter  is  stoppered  and  the  urine 
drawn  off  at  the  end  of  each  hour.  Other  patients  may  simply  be  allowed  to  urinate 
at  the  hourly  periods. 

To  each  hour  sample  of  urine  is  added  25  per  cent  NaOH,  drop  by  drop,  imtil 
the  mayimnm  intensity  of  color  appears.  This  color  will  remain  constant  for  an 
indefinite  period  of  time.  Each  sample  is  then  placed  in  a  1000  c.c.  volvunetric 
flask  and  diluted  to  the  mark  with  distilled  water. 

Comparison  is  made  in  a  Duboscq  (Hellige  or  Sargent)  colorimeter  (see  p.  486) 
with  a  standard  consisting  of  3  mg.  of  phenolsulphonephthalein  in  1000  c.c.  of  solu- 
tion. The  cylinder  containing  the  standard  may  conveniently  be  placed  at  the  10 
mm.  mark.  Since  the  volmne  of  each  urine  sample  is  the  same  as  that  of  the 
standard,  the  percentage  elimination  of  phenolsulphonephthalein  in  each  may  be 
easily  calculated  as  follows : 

Reading  of  Urine :  Reading  of  Standard : :  100 :  X. 

The  amount  of  the  drug  eliminated  normally  is  40-60  per  cent  during 
the  first  hour  and  20-25  per  cent  during  the  second  hour,  or  a  total  of 
60-85  P^r  cent  for  two  hours.  The  amount  of  the  drug  excreted  has 
been  found  to  be  independent  of  the  quantity  of  urine  obtained. 
In  case  of  delayed  excretion  the  collection  of  hourly  samples  may  be 
continued  until  practically  all  of  the  drug  has  been  recovered  in  the 
urine. 

If  it  is  desired  to  test  the  function  of  each  kidney  separately, 
ureteral  catheterization  must  be  resorted  to,  the  experiment  other- 
wise being  performed  as  above  described. 

The  phenolsulphonephthalein  test  may  be  used  to  indicate  the 
amount  of  derangement  in  quantitative  functional  disturbance  of  the 
kidneys,  as  in  chronic  interstitial  and  chronic  parenchymatous  neph- 
ritis or  uremia. 

McLean^  has  very  recently  suggested  a  method  for  studying  kidney 
function  which  is  based  upon  the  relationship  between  the  urea  con- 
tent of  the  blood  and  the  rate  at  which  the  urea  is  excreted  by  the 
kidney.  It  gives  similar  values  to  the  phenol-sulphonephthalein  test. 
It  has  an  advantage  in  that  it  enables  one  to  measure  kidney  function 
by  a  study  of  an  actual  normal  function  of  the  organ,  i.e.,  urea  excre- 
tion.    The  method,  however,  is  more  or  less  complex. 

^McLean:  Jour.  Am.  Med.  Assn.,  66.,  415,  1916. 


CHAPTER  XXIV 

URINE :  ORGANIZED  AND  UNORGANIZED 
SEDIMENTS 

The  data  obtained  from  carefully  conducted  microscopical  exami- 
nations of  the  sediment  of  certain  pathological  urines  are  of  very  great 
importance  diagnostically.  Too  little  emphasis  is  sometimes  placed 
upon  the  value  of  such  findings. 

The  sedimentary  constituents  may  be  divided  into  two  classes, 
i.e.,  organized  and  unorganized.     The  sediment  is  ordinarily  collected 


Fig.  132. — The  Purdy  Electric  Centripvge. 


Fig.  133. — Sediment  Tube  FOR  THE 
PcTRDY  Electric  Centrifvge. 


for  examination  by  means  of  the  centrifuge  (Fig.  132).  An  older 
method,  and  one  still  in  vogue  in  some  quarters,  is  the  so-called  gravity 
method.  This  simply  consists  in  placing  the  urine  in  a  conical  glass 
and  allowing  the  sediment  to  settle.  The  collection  of  the  sediment  by 
means  of  the  centrifuge,  however,  is  much  preferable,  since  the  process 
of  sedimentation  may  be  accomplished  by  the  use  of  this  instrument  in  a 
few  minutes,  and  far  more  perfectly,  whereas  when  the  other  method  is 
used  it  is  frequently  necessary  to  allow  the  urine  to  remain  in  the  con- 

457 


45^  PHYSIOLOGICAL   CHEMISTRY 

ical  glass  12-24  hours  before  sufficient  sediment  can  be  secured  for  the 
microscopical  examination. 

(a)  Unorganized  Sediments 

Ammonium  magnesium  phosphate  ("triple  phosphate")- 

Calcium  oxalate. 

Calcium  carbonate. 

Calcium  phosphate. 

Calcium  sulphate. 

Uric  acid. 

Urates. 

Cystine. 

Cholesterol. 

Hippuric  acid. 

Leucine  (?)  and  tyrosine. 

Hematoidin  and  bilirubin. 

Magnesium  phosphate. 

Indigo. 

Xanthine. 

Melanin. 

Ammonium  Magnesium  Phosphate  ("Triple  Phosphate"). — 
Crystals  of  "triple  phosphate"  are  a  characteristic  constituent  of  the 
sediment  when  alkaline  fermentation  of  the  urine  has  taken  place 
either  before  or  after  being  voided.  They  may  even  be  detected  in 
amphoteric  or  slightly  acid  urine  provided  the  ammonium  salts  are 
present  in  large  enough  quantity.  This  substance  may  occur  in  the 
sediment  in  two  forms,  i.e.,  prisms  and  the  feathery  type.  The  pris- 
matic form  of  crystals  (Fig.  129,  page  408)  is  the  one  most  commonly 
observed  in  the  sediment;  the  feathery  form  (Fig.  129,  page  408)  pre- 
dominates when  the  urine  is  made  ammoniacal  with  ammonia. 

The  sediment  of  the  urine  in  such  disorders  as  are  accompanied  by 
a  retention  of  urine  in  the  lower  urinary  tract  contains  "triple  phos- 
phate" crystals  as  a  characteristic  constituent.  The  crystals  are  fre- 
quently abundant  in  the  sediment  during  paraplegia,  chronic  cystitis, 
enlarged  prostate,  and  chronic  pyelitis. 

Calcium  Oxalate. — Calcium  oxalate  is  found  in  the  urine  in  the 
form  of  at  least  two  distinct  types  of  crystals,  i.e.,  the  dumb-bell  type 
and  the  octahedral  type  (Fig.  134,  page  459).  Either  form  may  occur 
in  the  sediment  of  neutral,  alkaline,  or  acid  urine,  but  both  forms  are 
found  most  frequently  in  urine  having  an  acid  reaction.  Occasionally, 
in  alkaline  urine,  the  octahedral  form  is  confounded  with  "triple  phos- 


URINE 


459 


phate"  crystals.     They  may  be  differentiated  from   the    phosphate 
crystals  by  the  fact  that  they  are  insoluble  in  acetic  acid. 

The  presence  of  calcium  oxalate  in  the  urine  is  not  of  itself  a  sign  of 
any  abnormality,  since  it  is  a  constituent  of  normal  urine.  It  is  increased 
above  the  normal,  however,  in  such  pathological  conditions  as  diabetes 


© 


^ 


# 


^ 


^ 


^ 


W^^       ^" 


Fig.  134. — Calcium  Oxalate.     (Ogden.) 

mellitus,  in  organic  diseases  of  the  liver,  and  in  various  other  conditions 
which  are  accompanied  by  a  derangement  of  digestion  or  of  the  oxida- 
tion mechanism,  such  as  occurs  in  certain  diseases  of  the  heart  and 
lungs. 

Calcium  Carbonate. — Calcium  carbonate  crystals  form  a  typical 
constituent  of  the  urine  of  herbivorous  animals.     They  occur  less  fre- 


FiG.  135. — Calcium  Carbonate. 

quently  in  human  urine.  The  reaction  of  urine  containing  these 
crystals  is  nearly  always  alkaline,  although  they  may  occur  in  ampho- 
teric or  in  slightly  acid  urine.  It  generally  crystallizes  in  the  form 
of  granules,  spherules,  or  dumb-bells  (Fig.  135).  The  crystals  of 
calcium  carbonate  may  be  differentiated  from  calcium  oxalate  bv  the 


460  PHYSIOLOGICAL   CHEMISTRY 

fact  that  they  dissolve  in  acetic  acid  with  the  evolution  of  carbon  dioxide 
gas. 

Calcium  Phosphate  (Stellar  Phosphate). — Calcium  phosphate  may- 
occur  in  the  urine  in  three  forms,  i.e.,  amorphous,  granular,  or  crystal- 
line. The  crystals  of  calcium  phosphate  are  ordinarily  pointed,  wedge- 
shaped  formations  which  may  occur  as  individual  crystals  or  grouped 
together  in  more  or  less  regularly  formed  rosettes  (Fig.  105,  page  322). 
Acid  sodium  urate  crystals  (Fig.  137,  page  462)  are  often  mistaken  for 
crystals  of  calcium  phosphate.  We  may  differentiate  between  these 
two  crystalline  forms  by  the  fact  that  acetic  acid  will  readily  dissolve 
the  phosphate,  whereas  the  urate  is  much  less  soluble  and  when  finally 
brought  into  solution  and  recrystallized  one  is  frequently  enabled  to 
identify  uric  acid  crystals  which  have  been  formed  from  the  acid  urate 
solution.  The  clinical  significance  of  the  occurrence  of  calcium  phos- 
phate crystals  in  the  urinary  sediment  is  similar  to  that  of  "triple 
phosphate"  (see  page  458). 

Calcium  Sulphate. — Crystals  of  calcium  sulphate  are  of  quite  rare 
occurrence  in  the  sediment  of  urine.  Their  presence  seems  to  be 
limited  in  general  to  urines  which  are  of  a  decided  acid  reaction. 
Ordinarily  it  crystallizes  in  the  form  of  long,  thin,  colorless  needles  or 
prisms  (Fig.  128,  page  405)  which  may  be  mistaken  for  calcium  phos- 
phate crystals.  There  need  be  no  confusion  in  this  respect,  however, 
since  the  sulphate  crystals  are  insoluble  in  acetic  acid,  which  reagent 
readily  dissolves  the  phosphate.  As  far  as  is  known  their  occurrence 
as  a  constituent  of  urinary  sediment  is  of  very  little  clinical  significance. 

Uric  Acid. — Uric  acid  forms  a  very  common  constituent  of  the  sedi- 
ment of  urines  which  are  acid  in  reaction.  It  occurs  in  more  varied 
forms  than  any  of  the  other  crystalline  sediments  (Plate  V,  opposite 
page  380,  and  Fig.  136),  some  of  the  more  common  varieties  of  crystals 
being  rhombic  prisms,  wedges,  dumb-bells,  whetstones,  prismatic 
rosettes,  irregular  or  hexagonal  plates,  etc.  Crystals  of  pure  uric  acid 
are  always  colorless  (Fig.  122,  page  380),  but  the  form  occurring  in 
urinary  sediments  is  impure  and  under  the  microscope  appears  pig- 
mented, the  depth  of  color  varying  from  yellow  to  a  dark  reddish- 
brown  according  to  the  size  and  form  of  the  crystal. 

The  presence  of  a  considerable  uric  acid  sediment  does  not,  of  neces- 
sity, indicate  a  pathological  condition  or  a  urine  of  increased  uric  acid 
content,  since  this  substance  very  often  occurs  as  a  sediment  in  urines 
whose  uric  acid  content  is  diminished  from  the  normal  merely  as  a  re- 
sult of  changes  in  reaction,  etc.  Pathologically,  uric  acid  sediments  oc- 
cur in  gout,  acute  febrile  conditions,  chronic  interstitial  nephritis,  etc. 
If  the  microscopical  examination  is  not  conclusive,  uric  acid  may  be 


"URINE 


461 


dififerentiated  from  other  crystalline  urinary  sediments  from  the  fact 
that  it  is  soluble  in  alkalis,  alkali  carbonates,  boiling  glycerol,  concen- 
trated sulphuric  acid,  and  in  certain  organic  bases  such  as  ethylamine 
and  piperidin.  It  also  responds  to  the  murexide  test  (see  page  380), 
Schiflf's  reaction  (see  page  381)  and  to  Folin's  phosphotungstic  acid 
reaction  (see  page  381). 

Urates. — The  urate  sediment  may  consist  of  a  mixture  of  the  urates 
of  ammonium,  calcium,  magnesium,  potassium,  and  sodium.  The 
ammonium  urate  may  occur  in  neutral,  alkaline,  or  acid  urine,  whereas 
the  other  forms  of  urates  are  confined  to  the  sediments  of  acid  urines. 
Sodium  urate  occurs  in  sediments  more  abundantly  than  the  other 


Fig.  136. — Various  Forms  of  Uric  Acid. 

I,  Rhombic  plates;  2,  whetstone  forms;  3,  3,  quadrate  forms;  4,  5,  prolonged  into 
points;  6,  8,  rosettes;  7,  pointed  bundles;  9,  barrel  forms  precipitated  by  adding  hydro- 
chloric acid  to  urine. 


urates.     There  are  two  sodium  urates,  the  mono  and  the  di,  which  may 

Na"'"\  _  Na'^X  _ 

be  expressed  thus    jj+  >C5H2N403     and  j^^+ yCsHaNiOs    .     Both 

salts  dissociate  with  the  production  of  an  alkaline  reaction,  the  alka- 
linity being  stronger  in  the  case  of  the  di-sodium  urate.  The  so-called 
quadriurate  or  hemiurate  have  no  existence  as  chemical  units.  ^  The 
urates  of  calcium,  magnesium,  and  potassium  are  amorphous  in 
character,  whereas  the  urate  of  ammonium  is  crystalline.  Sodium 
urate  may  be  either  amorphous  or  crystalline.  When  crystalline  it 
forms  groups  of  fan-shaped  clusters  or  colorless,  prismatic  needles  (Fig. 

*  Taylor:  Jour.  Biol.  Cliem.,  i,  177,  1905. 


462 


PHYSIOLOGICAL   CHEMISTRY 


137.  Ammonium  urate  is  ordinarily  present  in  the  sediment  in  the 
burr-like  form  of  the  "thorn-apple"  crystal,  i.e.,  yellow  or  reddish- 
brown  spheres,  covered  with  sharp  spicules  or  prisms  (Plate  VI, 
opposite).  The  urates  are  all  soluble  in  hydrochloric  acid  or  acetic 
acid  and  their  acid  solutions  yield  crystals  of  uric  acid  upon  standing. 
They  also  respond  to  the  murexide  test.     The  clinical  significance  of 


Fig.  137. — Acid  Sodium  Urate. 

urate  sediments  is  very  similar  to  that  of  uric  acid.  A  considerable 
sediment  of  amorphous  urates  does  not  necessarily  indicate  a  high  uric 
acid  content,  but  ordinarily  signifies  a  concentrated  urine  having  a  very 
strong  acidity. 

Cystine. — Cystine  is  one  of  the  rarer  of  the  crystalline  urinary  sedi- 
ments.    It  has  been  claimed  that  it  occurs  more  often  in  the  urine  of 


0 


# 


/ 


Fig.  138. — Cystine.     (Ogden.) 

men  than  of  women.  Cystine  crystallizes  in  the  form  of  thin,  color- 
less, hexagonal  plates  (Fig.  26,  page  76,  and  Fig.  138)  which  are 
insoluble  in  water,  alcohol,  and  acetic  acid,  and  soluble  in  minerals, 
acids,  alkalis,  and  especially  in  ammonia.  Cystine  may  be  identified 
by  burning  it  upon  platinum  foil,  under  which  condition  it  does  not 


PLATE  VI. 


Ammonium  Urates,  showing  Spherules  and  Thorx-apple-shaped  Crystals. 
(From  Ogden,  after  Pcycr.) 


URINE  463 

melt  but  yields  a  bluish-green  flame.  For  preparation  of  Cystine  see 
Chapter  IV. 

Cholesterol. — Cholesterol  crystals  have  been  but  rarely  detected  in 
urinary  sediments.  When  present  they  probably  arise  from  a  patho- 
logical condition  of  some  portion  of  the  urinary  tract.  Crystals  of 
cholesterol  have  been  found  in  the  sediment  in  cystitis,  pyelitis, 
chyluria,  and  nephritis.  Ordinarily  it  crystallizes  in  large  regular  and 
irregular  colorless,  transparent  plates,  some  of  which  possess  notched 
corners  (Fig.  57,  page  210).  Frequently,  instead  of  occurring  in  the 
sediment,  it  is  found  in  the  form  of  a  film  on  the  surface  of  the  urine. 

Hippuric  Acid. — This  is  one  of  the  rare  sediments  of  human  urine. 
It  deposits  under  conditions  similar  to  those  which  govern  the  formation 
of  uric  acid  sediments.  The  crystals, 
which  are  colorless  needles  or  prisms 
(Fig.  125,  page  389)  when  pure,  are  in- 
variably pigmented  in  a  manner  similar 
to  the  uric  acid  crystals  when  observed 
in  urinary  sediment  and  because  of  this 

fact  are  frequently  confounded  with  the     \ff^  .^jfa^ 

rarer  forms  of  uric  acid.     Hippuric  acid  ^©  ^Qf 

may   be   differentiated  from  uric  acid  ^ 

from  the  fact  that  it  does  not  respond  to       Fig.  139.— Crystals  of  Impure 

Leucixe.     (Ogden.) 
the  murexide  test  and  is  much  more 

soluble  in  water  and  in  ether.  The  detection  of  crystals  of  hippuric 
acid  in  the  urine  has  very  little  chnical  significance,  since  its  pres- 
ence in  the  sediment  depends  in  most  instances  very  greatly  upon 
the  nature  of  the  diet.  It  is  particularly  prone  to  occur  in  the  sedi- 
ment after  the  ingestion  of  certain  fruits  as  well  as  after  the  ingestion  of 
benzoic  acid  (see  pages  388  and  585). 

Leucine  and  Tyrosine. — ^Leucine  and  tyrosine  have  frequently 
been  detected  in  the  urine,  either  in  solution  or  as  a  sediment.  Neither 
of  them  occurs  in  the  urine  ordinarily  except  in  association  with  the 
other,  i.e.,  whenever  leucine  is  detected  it  is  more  than  probable  that 
tyrosine  accompanies  it.  They  have  been  found  pathologically  in 
the  urine  in  acute  yellow  atrophy  of  the  liver,  in  acute  phosphorus 
poisoning,  in  cirrhosis  of  the  liver,  in  severe  cases  of  typhoid  fever 
and  small-pox,  and  in  leukemia.  In  urinary  sediments  leucine  ordi- 
narily crystallizes  in  characteristic  spherical  masses  which  show  both 
radial  and  concentric  striations  and  are  highly  refractive  (Fig.  139). 
Some  investigators  claim  that  these  crystals  which  are  ordinarily  called 
leucine  are,  in  reality,  generally  urates.  This  view  point  has  become 
more  general  in  recent  years.     For  the  crystalline  form  of  pure  leucine 


464  PHYSIOLOGICAL    CHEMISTRY 

obtained  as  a  decomposition  product  of  protein  see  Fig.  28,  page  80. 
Tyrosine  crystallizes  in  urdinary  sediments  in  the  well-known  sheaf 
or  tuft  formation  (Fig.  25,  page  76).  For  other  tests  on  leucine  and 
tyrosine  see  pages  86  and  87. 

Hematoidin  and  Bilirubin. — There  are  divergent  opinions  regard- 
ing the  occurrence  of  these  bodies  in  urinary  sediment.  Each  of  them 
crystallizes  in  the  form  of  tufts  of  small  needles  or  in  the  form  of  small 
plates  which  are  ordinarily  yellowish-red  in  color  (Fig.  56,  page  205). 
Because  the  of  fact  that  the  crystalline  form  of  the  two  substances  is 
identical  many  investigators  claim  them  to  be  one  and  the  same  body. 
Other  investigators  claim,  that  while  the  crystalline  form  is  the  same 
in  each  case,  there  are  certain  chemical  differences  which  may  be  brought 
out  very  strikingly  by  properly  testing.  For  instance,  it  has  been 
claimed  that  hematoidin  may  be  differentiated  from  bilirubin  through 
the  fact  that  it  gives  a  momentary  color  reaction  (blue)  when  nitric  acid 
is  brought  into  contact  with  it,  and,  further,  that  it  is  not  dissolved  on 
treatment  with  ether  or  potassium  hydroxide.  Pathologically,  typical 
crystals  of  hematoidin  or  bilirubin  have  been  found  in  the  urinary 
sediment  in  jaundice,  acute  yellow  atrophy  of  the  liver,  carcinoma  of 
the  liver,  cirrhosis  of  the  liver,  and  in  phosphorus  poisoning,  typhoid 
tever,  and  scarlatina. 

Magnesium  Phosphate. — Magnesium  phosphate  crystals  occur 
rather  infrequently  in  the  sediment  of  urine  which  is  neutral,  alkaline, 
or  feebly  acid  in  reaction.  It  ordinarily  crystallizes  in  elongated, 
highly  refractive,  rhombic  plates  which  are  soluble  in  acetic  acid. 

Indigo. — Indigo  crystals  are  frequently  found  in  urine  which  has 
undergone  alkaline  fermentation.  They  result  from  the  breaking 
down  of  indoxyl-sulphates  or  indoxyl-glycuronates.  Ordinarily  indigo 
deposits  as  dark  blue  stellate  needles  or  occurs  as  amorphous  particles 
or  broken  fragments.  These  crystalline  or  amorphous  forms  may  occur 
in  the  sediment  or  may  form  a  blue  film  on  the  surface  of  the  urine. 
Indigo  crystals  generally  occur  in  urine  which  is  alkaline  in  reaction, 
but  they  have  been  detected  in  acid  urine. 

Xanthine. — Xanthine  is  a  constituent  of  normal  urine  but  is  found 
in  the  sediment  in  crystalline  form  very  infrequently,  and  then  only  in 
pathological  urine.  When  present  in  the  sediment  xanthine  generally 
occurs  in  the  form  of  whetstone-shaped  crystals  somewhat  similar  in 
form  to  the  whetstone  variety  of  uric  acid  crystal.  They  may  be  dif- 
ferentiated from  uric  acid  by  the  great  ease  with  which  they  may  be 
brought  into  solution  in  dilute  ammonia  and  on  applying  heat.  Xan- 
thine may  also  form  urinary  calculi.  The  clinical  significance  of 
xanthine  in  urinarv  sediment  is  not  well  understood. 


URIISTE  465 

Melanin. — Melanin  is  an  extremely  rare  constituent  of  urinary 
sediments.  Ordinarily  in  melanuria  the  melanin  remains  in  solution; 
if  it  separates  it  is  generally  held  in  suspension  as  fine  amorphous 
granules. 

(b)  Organized  Sediments 

Epithelial  cells. 

Pus  cells. 

Hyaline. 

Granular. 

Epithelial. 

Casts.        ^  Blood. 
Fatty. 
Waxy. 
.  Pus. 

Cyhndroids.  '.  '  ' 

Erythrocytes. 

Spermatozoa. 

Urethral  filaments. 

Tissue  debris. 

Animal  parasites. 

Micro-organisms. 

Fibrin. 

Foreign  substances  due  to  contamination. 

Epithelial  Cells. — The  detection  of  a  certain  number  of  these  cells 
in  urinary  sediment  is  not,  of  itself,  a  pathological  sign,  since  they 
occur  in  normal  urine.  However,  in  certain  pathological  conditions 
they  are  greatly  increased  in  number,  and  since  different  areas  of  the 
urinary  tract  are  lined  with  different  forms  of  epithelial  cells,  it  becomes 
necessary,  when  examining  urinary  sediments,  to  note  not  only  the 
relative  number  of  such  cells,  but  at  the  same  time  to  carefully  observe 
the  shape  of  the  various  individuals  in  order  to  determine,  as  far  as 
possible,  from  what  portion  of  the  tract  they  have  been  derived.  Since 
the  different  layers  of  the  epithelial  lining  are  composed  of  cells  dif- 
ferent in  form  from  those  of  the  associated  layers,  it  is  evident  that  a 
careful  microscopical  examination  of  these  cells  may  tell  us  the  par- 
ticular layer  which  is  being  desquamated.  It  is  frequently  a  most  diffi- 
cult undertaking,  however,  to  make  a  clear  differentiation  between  the 
various  forms  of  epithelial  cells  present  in  the  sediment.  If  skilfully 
done,  such  a  microscopical  differentiation  may  prove  to  be  of  very 
great  diagnostic  aid. 

The  principal  forms  of  epithelial  cells  met  with  in  urinary  sediments 
are  shown  in  Fig.  140,  page  466. 


466 


PHYSIOLOGICAL    CHEMISTRY 


Pus  Cells.- — Pus  corpuscles  or  leucocytes  are  present  in  extremely 
small  numbers  in  normal  urine.  Any  considerable  increase  in  the 
number,  however,  ordinarily  denotes  a  pathological  condition,  gener- 
ally an  acute  or  chronic  inflammatory  condition  of  some  portion  of  the 
urinary  tract.  The  sudden  appearance  of  a  large  amount  of  pus  in  a 
sediment  denotes  the  opening  of  an  abscess  into  the  urinary  tract.  Other 
form  elements,  such  as  epithelial  cells,  casts,  etc.,  ordinarily  accompany 
pus  corpuscles  in  urinary  sediment  and  a  careful  examination  of  these 
associated  elements  is  necessary  in  order  to  form  a  correct  diagnosis  as 
to  the  origin  of  the  pus.  Protein  is  always  present  in  urine  which 
contains  pus. 


Fig.  140. — Epithelium  from  Different  Areas  of  the  Urinary  Tract. 

a,  Leucocyte  (for  comparison);  b,  renal  cells;  c,  superficial  pelvic  cells;  d,  deep  pelvic 
cells;  e,  cells  from  calices;  /,  cells  from  ureter;  g,  g,  g,  g,  g,  squamous  epithelium  from  the 
bladder;  h,  h,  neck-of-bladder  cells;  i,  epithelium  from  prostatic  urethra;  k,  urethral  cells; 
/,  /,  scaly  epithelium;  m,  m',  cells  from  seminal  passages;  n,  compound  granule  cells;  0, 
fatty  renal  cell.     (Ogden.) 

The  appearance  which  pus  corpuscles  exhibit  under  the  microscope 
depends  greatly  upon  the  reaction  of  the  urine  containing  them.  In 
acid  urine  they  generally  present  the  appearance  of  round,  colorless  cells 
composed  of  refractive,  granular  protoplasm,  and  may  frequently  exhibit 
ameboid  movements,  especially  if  the  slide  containing  them  be  warmed 
slightly.  They  are  nucleated  (one  or  more  nuclei),  the  nuclei  being 
clearly  visible  only  upon  treating  the  cells  with  water,  acetic  acid,  or 
some  other  suitable  reagent.  In  urine  which  has  a  decided  alkaline 
reaction,  on  the  other  hand,  the  pus  corpuscles  are  often  greatly  de- 
generated. They  may  be  seen  as  swollen,  transparent  cells,  which 
exhibit  no  granular  structure  and  as  the  process  of  degeneration  con- 


URIXE. 


467 


tinues  the  cell  outline  ceases  to  be  visible,  the  nuclei  fade,  and  finally 
only  a  mass  of  debris  containing  isolated  nuclei  and  an  occasional 
cell  remains. 

It  is  frequently  rather  difficult  to  make  a  differentiation  between  pus 
corpuscles  and  certain  types  of  epithelial  cells  which  are  similar  in  form. 
Such  confusion  may  be  avoided  by  the  addition  of  iodine  solution  (I  in 
KI),  a  reagent  which  stains  the  pus  corpuscles  a  deep  mahogany-brown 
and  transmits  to  the  epithelial  cells  a  light  yellow  tint.  The  test  pro- 
posed by  Vitali  often  gives  very  satisfactory  results.  This  simply 
consists  in  acidifying  the  urine  fif  alkaline)  with  acetic  acid,  then  filter- 
ing, and  treating  the  sediment  on  the  filter  paper  with  freshly  prepared 
tincture  of  guaiac.     The  presence  of  pus  in  the  sediment  is  indicated 


Fig.  141. — Pus  Corpuscles.     (After  Ullzmann.) 

I,  Normal;  2,  showing  amoeboid  movements;  3,  nuclei  rendered  distinct  by  acetic  acid;  4, 
as  observed  in  chronic  pyelitis;  5,  swollen  by  ammonium  carbonate. 


if  a  blue  color  is  observed.  Large  numbers  of  pus  corpuscles  are  present 
in  the  urinary  sediment  in  gonorrhoea,  leucorrhcra,  chronic  pyelitis, 
and  in  abscess  of  the  kidney.  In  addition  to  the  usual  constituents 
found  in  leucocytes  Mandel  and  Levene^  claim  that  pus  cells  contain 
glucolhionic  acid.     See  Pus  tests,  page  431. 

Casts. — These  are  cylindrical  formations,  which  originate  in  the 
uriniferous  tubules  and  are  forced  out  by  the  pressure  of  the  urine. 
They  vary  greatly  in  size,  but  in  nearly  every  instance  they  possess 
parallel  sides  and  rounded  ends.  The  finding  of  casts  in  the  urine  is 
very  important  because  of  the  fact  that  they  generally  indicate  some 
kidney  disorder;  if  albumin  accompanies  the  casts  the  indication  is 

'  Mandel  and  Levene:  Biochemischc  Zeitschrift,  4,  jH,  1007. 


468 


PHYSIOLOGICAL    CHEMISTRY 


much  accentuated.  Casts  have  been  classified  according  to  their 
microscopical  characteristics  as  follows:  (a)  hyaline,  {b)  granular,  (c) 
epithelial,  (d)  blood,  {e)  fatty,  (/)  waxy,  (g)  pus. 

(fl)  Hyaline  Casts. — These  are  composed  of  a  basic  material  which 
is  transparent,  homogeneous,  and  very  light  in  color  (Fig.  142). 
In  fact,  chiefly  because  of  these  physical  properties,  they  are  the 
most  difl&cult  form  of  renal  casts  to  detect  under  the  microscope. 
Frequently  such  casts  are  impregnated  with  deposits  of  various  forms, 
such  as  erythrocytes,  epithelial  cells,  fat  globules,  etc.,  thus  rendering 
the  form  of  the  cast  more  plainly  visible.     Staining  is  often  resorted  to 


Fig.  142. — Hyaline  Casts. 
One  cast  is  impregnated  with  four  renal  cells. 


in  order  to  render  the  shape  and  character  of  the  cast  more  easily 
determined.  Ordinary  iodine  solution  (I  in  KI)  may  be  used  in  this 
connection;  many  of  the  aniline  dyes  are  also  in  common  use  for  this 
purpose,  e.g.,  gentian- violet,  Bismarck-brown,  methylene-blue,  fuchsin, 
and  eosin.  Generally,  but  not  always,  albumin  is  present  in  urine 
containing  hyaline  casts.  Hyaline  casts  are  common  to  all  kidney 
disorders,  but  occur  particularly  in  the  earliest  and  recovering  stages 
of  parenchymatous  nephritis  and  interstitial  nephritis. 

{b)  Granular  Casts. — The  common  hyaline  material  is  ordinarily  the 
basic  substance  of  this  form  of  cast.  The  granular  material  generally 
consists  of  albumin,  epitheial  cells,  fat,  or  disintegrated  erythrocytes  or 


URIXE 


469 


leucocytes,  the  character  of  the  cast  varjdng  according  to  the  nature 
and  size  of  the  granules  (Fig.  143,  and  Fig.  144,  page  470J.  Thus 
we  have  casts  of  this  general  type  classified  as  finely  granular  and 
coarsely  granular  casts.  Granular  casts,  and  in  particular  the  finely 
granular  types,  occur  in  the  sediment  in  practically  every  kidney  dis- 
order but  are  probably  especially  characteristic  of  the  sediment  in  in- 
flammatory disorders. 

{c)  Epithelial  Casts. — These  are  casts  bearing  upon  their  surface 
epithelial  cells  from  the  lining  of  the  uriniferous  tubules  (Fig.  145, 
page  470).     The  basic  material  of  this  form  of  cast  may  be  hyaline  or 


Fig.  143. — Granular  Casts.     (After  Peyer.) 


granular  in  nature.     Epithelial  casts  are  particularly  abundant  in  the 
urinary  sediment  in  acute  nephritis. 

(d)  Blood  Casts. — Casts  of  this  type  may  consist  of  erythrocytes 
borne  upon  a  hyaline  or  a  fibrinous  basis  (Fig.  146,  page  470).  The 
occurrence  of  such  casts  in  the  urinary  sediment  denotes  renal  hemor- 
rhage and  they  are  considered  to  be  especially  characteristic  of  acute 
diffuse  nephritis  and  acute  congestion  of  the  kidney. 

(e)  Fatty  Casts. — Fatty  casts  may  be  formed  by  the  deposition  of 
fat  globules  or  crystals  of  fatty  acid  upon  the  surface  of  a  hyaline  or 
granular  cast  (Fig.  147,  page  471).  In  order  to  constitute  a  true  fatty 
cast  the  deposited  material  must  cover  the  greater  part  of  the  surface 
area  of  the  cast.     The  presence  of  fatty  casts  in  urinary  sediment  in- 


470 


PHYSIOLOGICAL    CHEMISTRY 


dicatesjatty  degeneration  of  the  kidney;  such  casts  are  particularly 
characteristic  of  subacute  and  chronic  inflammation  of  the  kidney. 


Fig.  144. — Granular  Casts. 
a  Finely  granular;    b,  coarsely  granular. 


Fig.  145. — Epithell^l  Casts. 


(/)  Waxy  Casts. — These  casts  possess  a  basic  substance  similar  to 
that-which  enters  into  the  foundation  of  the  hvaline  form  of  cast.     In 


Fig.  146. — Blood,  Pus,  Hyaline  and  Epithellil  Casts. 
a,  Blood  casts;  b,  pus  cast;  c,  hyaline  cast  impregnated  with  renal  cells;  d,  epithelial  casts. 

common  with  the  hyaline  type  they  are  colorless,  refractive  bodies, 
but  diflfer  from  this  form  of  cast  in  being,  in  general,  of  greater  length 
and  diameter  and  possessing  sharper  outlines  and  a  light  yellow  color 


URINE 


471 


Fig.  147. — Fatty  Casts.     {Aitei  Peyer.) 


Fig.  148. — Fatty  and  Waxy  Casts. 
a,  Fatty  casts;  b,  waxy  casts. 


472 


PHYSIOLOGICAL   CHEMISTRY 


(Fig.  148,  page  471).  Such  casts  occur  in  several  forms  of  nephritis, 
but  do  not  appear  to  characterize  any  particular  type  of  the  disorder 
except  amyloid  disease,  in  which  they  are  rather  common. 

(g)  Pus  Casts. — Casts  whose  surface  is  covered  with  pus  cells  or 
leucocytes  are  termed  pus  casts  (Fig.  146,  p.  470).  They  are  frequently 
mistaken  for  epithehal  casts.  The  differentiation  between  these  two 
types  is  made  very  simple,  however,  by  treating  the  cast  with  acetic 
acid  which  causes  the  nuclei  of  the  leucocytes  to  become  plainly  visible. 
The  true  pus  cast  is  quite  rare  and  indicates  renal  suppuration. 

Cylindroids. — These  formations  may  occur  in  normal  or  pathological 
urine  and  have  no  particular  clinical  significance.     They  are  frequently 


Fig.  149. — CvLiNDRorDS.     {Mter  Peyer.) 

mistaken  for  true  casts,  especially  the  hyaline  type,  but  they  are 
ordinarily  flat  in  structure  with  a  rather  smaller  diameter  than  casts, 
may  possess  forked  or  branching  ends,  and  are  not  composed  of  homo- 
geneous material  as  are  the  hyaline  casts.  Such  ''false  casts"  may 
become  coated  with  urates,  in  which  event  they  appear  granular  in 
structure.  The  basic  substance  of  cylindroids  is  often  the  nucleo- 
protein  of  the  urine  (Fig.  149,  above). 

Erythrocytes. — These  form  elements  are  present  in  the  urinary 
sediment  in  various  diseases.  They  appear  as  the  normal  biconcave, 
yellow  erythrocyte  (Plate  IV,  opposite  page  249)  or  may  exhibit  certain 
modifications  in  form,  such  as  the  crenated  type  (Fig.  150) 
which  is  often  seen  in  concentrated  urine.     Under  different  condi- 


URINE 


473 


tions  they  may  become  swollen  sufl&ciently  to  entirely  erase  the  bi- 
concave appearance  and  may  even  occur  in  the  form  of  colorless  spheres 
having  a  smaller  diameter  than  the  original  disc-shaped  corpuscles. 
Erythrocytes  are  found  in  urinary  sediment  in  hemorrhage  of  the 
kidney  or  of  the  urinary  tract,  in  traumatic  hemorrhage,  hemorrhage 
from  congestion,  and  in  hemorrhagic  diathesis. 

Spermatozoa. — Spermatozoa  may  be  detected  in  the  urinary  sedi- 
ment in  diseases  of  the  genital  organs,  as  well  as  after  coitus,  nocturnal 
emissions,  epileptic,  and  other  convulsive  attacks,  and  sometimes  in 
severe  febrile  disorders,  especially  in  typhoid  fever.  In  form  they  con- 
sist of  an  oval  body,  to  which  is  attached  a  long,  delicate  tail  (Fig. 


Fig.  150. — Crenated  Erythrocytes. 


151,  page  474).  Upon  examination  they  may  show  motility  or  may  be 
motionless. 

Urethral  Filaments. — These  are  peculiar  thread-like  bodies  which 
are  sometimes  found  in  urinary  sediment.  They  may  occasionally  be 
detected  in  normal  urine  and  pathologically  are  found  in  the  sediment  in 
acute  and  chronic  gonnorrhcea  and  in  urethrorrhcea.  The  ground-sub- 
stance of  these  urethral  filaments  is,  in  part  at  least,  similar  to  that  of  the 
cylindroids  (see  page  47  2) .  The  urine  first  voided  in  the  morning  is  best 
adapted  for  the  examination  for  filaments.  These  filaments  may  ordi- 
narily be  removed  by  a  pipette  since  they  are  generally  macroscopic. 

Tissue  Debris. — Masses  of  cells  or  fragments  of  tissue  are  frequently 
found  in  the  urinary  sediment.  They  may  be  found  in  the  sediment  in 
tubercular  affections  of  the  kidney  and  urinary  tract  or  in  tumors  of 
these  organs.     Ordinarily  it  is  necessary  to  make  a  histological  ex- 


474 


PHYSIOLOGICAL    CHEMISTRY 


amination  of  such  tissue  fragments  before  coining  to  a  final  decision  as 
to  their  origin. 

Animal  Parasites. — The  cysts,  hooklets,  and  membrane  shreds 
of  echinococci  are  sometimes  found  in  the  urinary  sediments.  Other 
animal  organisms  which  are  more  rarely  met  with  in  the  urine  are  em- 
bryos of  the  Filaria  sanguinis  and  eggs  of  the  Distoma  hematohinm  and 
Ascarides.  Animal  parasites  in  general  occur  most  frequently  in  the 
urine  in  tropical  countries. 

Micro-organisms. — Bacteria  as  well  as  yeasts  and  moulds  are  fre- 
quently detected  in  the  urine.  Both  the  pathogenic  and  non-patho- 
genic forms  of  bacteria  may  occur.  The  non-pathogenic  forms  most 
frequently  observed  are  micrococcus  urecB,  bacillus  urecB,  and  staphylo- 
coccus urece  liquefaciens.     Of  the  pathogenic  forms  many  have  been 


Fig.  151. — Human  Spermatozoa. 

observed,  e.g.,  Bacterium  Coli,  typhoid  bacillus,  tubercle  bacillus,  gono- 
coccus,  bacillus  pyocyaneus,  and  proteus  vulgaris.  Yeast  and  moulds 
are  most  frequently  met  with  in  diabetic  urine. 

Fibrin. — Following  hematuria,  fibrin  clots  are  occasionally  ob- 
served in  the  urinary  sediment.  They  are  generally  of  a  semi-gelatin- 
ous consistency  and  of  a  very  light  color,  and  when  examined  under 
the  microscope  they  are  seen  to  be  composed  of  bundles  of  highly  re- 
fractive fibers  which  run  parallel. 

Foreign  Substances  Due  to  Contamination.^ — Such  foreign  sub- 
stances as  fibers  of  silk,  linen,  or  wool;  starch  granules,  hair,  fat,  and 
sputum,  as  well  as  muscle  fibers,  vegetable  cells,  and  food  particles,  are 
often  found  in  the  urine.  Care  should  be  taken  that  these  foreign 
substances  are  not  mistaken  for  any  of  the  true  sedimentary  con- 
stituents already  mentioned. 


CHAPTER  XXV 
URINE:  CALCULI 

Urinary  calculi,  also  called  concretions,  or  concrements  are  solid 
masses  of  urinary  sediment  formed  in  some  part  of  the  urinary  tract. 
They  vary  in  shape  and  size  according  to  their  location,  the  smaller 
calculi,  termed  sand  or  gravel,  in  general  arising  from  the  kidney  or  the 
pelvic  portion  of  the  kidney,  whereas  the  large  calculi  are  ordinarily 
formed  in  the  bladder.  There  are  two  general  classes  of  calculi  as 
regards  composition,  i.e.,  simple  and  compound.  The  simple  form  is 
made  up  of  but  a  single  constituent,  whereas  the  compound  type  con- 
tains two  or  more  individual  constituents.  The  structural  plan  of 
most  calculi  consists  of  an  arrangement  of  concentric  rings  about  a 
central  nucleus,  the  number  of  rings  frequently  being  dependent  upon 
the  number  of  individual  constituents  which  enter  into  the  structure 
of  the  calculus.  However,  layers  quite  different  in  macroscopical 
appearance  may  be  almost  identical  in  composition.  In  case  two  or 
more  calculi  unite  to  form  a  single  calculus  the  resultant  body  %vill 
obviously  contain  as  many  nuclei  as  there  were  individual  calculi 
concerned  in  its  construction.  Under  certain  conditions  the  growth  of 
a  calculus  will  be  principally  in  only  one  direction,  thus  preventing 
the  nucleus  from  maintaining  a  central  location.  The  qualitative 
composition  of  urinary  calculi  is  dependent,  in  great  part,  upon  the 
reaction  of  the  urine,  e.g.,  if  the  reaction  of  the  urine  is  acid  the  calculi 
present  will  be  composed,  in  great  part  at  least,  of  substances  that  are 
capable  of  depositing  in  acid  urine,  e.g.,  uric  acid,  urates  and  calcium 
oxalates. 

According  to  Ultzmann,  out  of  545  cases  of  urinary  calculus,  uric 
acid  and  urates  formed  the  nucleus  in  about  81  per  cent  of  the  cases; 
earthy  phosphates  in  about  9  per  cent;  calcium  oxalate  in  about  6  per 
cent;  cystine  in  something  over  i  per  cent,  while  in  about  3  per  cent 
of  the  cases  some  foreign  body  comprised  the  nucleus. 

More  recent  analyses^  of  twenty-four  calculi  showed  the  nucleus  in 
75  per  cent  of  them  to  be  calcium  oxalate  (60  per  cent)  and  in  25  per 
cent  to  be  phosphate  (56  per  cent).  All  of  the  calculi  contained  some 
uric  acid  and  urates,  but  only  three  gave  more  than  10  per  cent. 

^  Kahn  and  Roscnbloom:  J  our.  Am.  Med.  Ass'n,  59,  2252,  1913. 

475 


476  PHYSIOLOGICAL   CHEMISTRY 

In  the  chemical  examination  of  urinary  calculi  the  most  valuable 
data  are  obtained  by  subjecting  each  of  the  concentric  layers  of  the 
calculus  to  a  separate  analysis.  Material  for  examination  may  be 
conveniently  obtained  by  sawing  the  calculus  carefully  through  the 
nucleus,  then  separating  the  various  layers,  or  by  scraping  off  from 
each  layer  (without  separating  the  layers)  enough  powder  to  conduct 
the  examination  as  outlined  in  the  scheme  (see  page  477). 

Varieties  of  Calculus 

Uric  Acid  and  Urate  Calculi. — Uric  acid  and  urates  constitute  the 
nuclei  of  a  large  proportion  of  urinary  concretions.  Such  stones  are 
always  colored,  the  tint  varying  from  a  pale  yellow  to  a  brownish-red. 
The  surface  of  such  calculi  is  generally  smooth  but  it  may  be  rough 
and  uneven. 

Phosphatic  Calculi.— Ordinarily  these  concretions  consist  prin- 
cipally of  "triple  phosphate"  and  other  phosphates  of  the  alkaline 
earths,  with  very  frequent  admixtures  of  urates  and  oxalates.  The 
surface  of  such  calculi  is  generally  rough  but  may  occasionally  be  rather 
smooth.  The  calculi  are  somewhat  variable  in  color,  exhibiting  gray, 
white,  or  yellow  tints  under  different  conditions.  When  composed  of 
earthy  phosphates  the  calculi  are  characterized  by  their  friability. 

Calcium  Oxalate  Calculi.— This  is  the  hardest  form  of  calculus 
to  deal  with,  and  is  rather  difficult  to  crush.  They  ordinarily  occur 
in  two  general  forms,  i.e.,  the  small,  smooth  concretion  which  is  char- 
acterized as  the  hemp-seed  calculus,  and  the  medium-sized  or  large  stone 
possessing  an  extremely  uneven  surface,  which  is  generally  classed  as 
a  mulberry  calculus.  This  roughened  surface  of  the  latter  form  of 
calculus  is  due,  in  many  instances,  to  protruding  calcium  oxalate 
crystals  of  the  octahedral  type. 

Calcium  Carbonate  Calculi.^ — Calcium  carbonate  concretions  are 
quite  common  in  herbivorous  animals,  but  of  exceedingly  rare  occur- 
rence in  man.  They  are  generally  small,  white,  or  grayish  calculi, 
spherical  in  form  and  possess  a  hard,  smooth  surface. 

Cystine  Calculi. — The  cystine  calculus  is  a  rare  variety  of  calculus. 
Ordinarily  they  occur  as  small,  smooth,  oval,  or  cylindrical  concretions 
which  are  white  or  yellow  in  color  and  of  a  rather  soft  consistency. 

Xanthine  Calculi. — This  form  of  calculus  is  somewhat  more  rare 
than  the  cystine  type.  The  color  may  vary  from  white  to  brownish- 
yellow.  Very  often  uric  acid  and  urates  are  associated  with  xanthine 
in  this  type  of  calculus.  Upon  rubbing  a  xanthine  calculus  it  has  the 
property  of  assuming  a  wax-like  appearance. 


URINE 


477 


On  Heating  the  Powder  on  Platinum  Foil,  It 


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478  PHYSIOLOGICAL    CHEMISTRY 

Urostealith  Calculi. — This  form  of  calculus  is  extremely  rare. 
Such  concretions  are  composed  principally  of  fat  and  fatty  acid.  When 
moist  they  are  soft  and  elastic,  but  when  dried  they  become  brittle. 
Urostealiths  are  generally  light  in  color. 

Fibrin  Calculi. — Fibrin  calculi  are  produced  in  the  process  of  blood 
coagulation  within  the  urinary  tract.  They  frequently  occur  as  nuclei 
of  other  forms  of  calculus.     They  are  rarely  found. 

Cholesterol  Calculi. — An  extremely  rare  form  of  calculus  somewhat 
resembling  the  cystine  type. 

Indigo  Calculi. — Indigo  calculi  are  extremely  rare,  only  two  cases 
having  been  reported.  One  of  these  indigo  calculi  is  on  exhibition  in 
the  museum  of  Jefferson  Medical  College  of  Philadelphia. 

The  scheme,  proposed  by  Heller  and  given  on  page  477,  will  be 
found  of  much  assistance  in  the  chemical  examination  of  urinary  calculi. 


CHAPTER  XXVI 

URINE :  QUANTITATIVE  ANALYSIS 

In  analyzing  a  normal  or  pathological  urine  quantitatively  for 
any  of  its  constituents  it  is  particularly  necessary  that  the  complete 
and  exact  24-hour  sample  be  obtained.  For  directions  with  regard  to 
the  collection  and  preservation  of  urine  for  analysis  see  Chapter 
XXI  on  General  Characteristics  of  Normal  and  Pathological  Urine. 
Methods  for  the  determination  of  the  specific  gravity  of  the  urine  are 
also  there  described.  Before  any  urine  is  taken  for  analysis  its  total 
volume  should  be  measured,  using  a  large  graduated  cylinder,  and  this 
volume  is  thereafter  taken  as  a  basis  for  the  calculations  of  the  daily 
output  of  the  individual  constituents  determined.  If  the  urine  be 
pathological  it  is  of  course  necessary  to  precede  its  quantitative 
analysis  by  qualitative  tests  for  the  pathological  constituents. 

Acidity  by  Titration 

Folin's  Method. — Principle: — The  urine  is  titrated  with  standard 
sodium  hydroxide  solution,  using  phenolphthalein  as  an  indicator. 
Potassium  oxalate  is  added  to  precipitate  the  calcium  which  would 
otherwise  interfere  with  the  end-point  due  to  the  precipitation  of  calcium 
phosphate  on  neutralization  of  the  urine.  The  acidity  of  the  urine  as 
determined  in  this  way  is  not  a  correct  measure  of  the  true  acidity,  which 
is  dependent  upon  the  concentration  of  hydrogen  ions.  The  results 
obtained  do,  however,  ordinarily  show  a  certain  parallelism  with  the 
hydrogen  ion  concentration  and  are  of  value  for  comparative  purposes. 

Procedure. — Place  25  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer  flask  and  add 
15-20  grams  of  finely  pulverized  potassium  oxalate  and  1-2  drops  of  a  i  per  cent 
phenolphthalein  solution  to  the  fluid.  Shake  the  mixture  vigorously  for  1-2 
minutes  and  titrate  it  immediately  with  N  10  sodiimi  hydroxide  until  a  faint  but 
unmistakable  pink  remains  permanent  on  further  shaking.  Take  the  burette 
reading  and  calculate  the  acidity  of  the  urine  under  examination. 

Calculation.  -  If  y  represents  the  number  of  cubic  centimeters  of  N  10  sodium 
hydroxide  used  and  y'  represents  the  volume  of  urine  excreted  in  24  hours,  the 
total  acidity  of  the  24-hour  urine  specimen  may  be  calculated  by  means  of  the 
following  proportion : 

479 


480  PHYSIOLOGICAL   CHEMISTRY 

25 :  y : :  y' :  X  (acidity  of  24-hour  urine  expressed  in  cubic  centimeters  of  N/io 

sodium  hydroxide). 

Each  cubic  centimeter  of  N/io  sodium  hydroxide  contains  0.004  gram  of 
sodium  hydroxide,  and  this  is  equivalent  to  0.0063  gram  of  oxaUc  acid.  There- 
fore, in  order  to  express  the  total  acidity  of  the  24-hour  urine  specimen  in  equiva- 
lent grams  of  sodium  hydroxide,  multiply  the  value  of  x,  as  just  determined, 
by  0.004,  or  multiply  the  value  of  x  by  0.0063  if  it  is  desired  to  express  the  total 
acidity  in  grams  of  oxaUc  acid. 

Interpretation. — (Under  the  heading  ''Interpretation^^  there  will 
be  found,  in  connection  with  the  various  quantitative  methods  which 
follow,  brief  notes  as  to  the  possible  significance  of  the  results  ob- 
tained. For  some  further  points  (and  reference  to  literature)  see  the 
chapters  on  the  Normal  and  Pathological  Constituents  of  Urine  and 
on  Metabolism.  Consult  text-books  on  physiological  chemistry  and 
clinical  diagnosis  for  complete  discussion).  The  acidity  of  the  urine 
expressed  in  cubic  centimeters  N/io  alkali  required  to  neutralize  the 
24-hour  output  varies  ordinarily  from  200  to  500  under  normal  con- 
ditions with  an  average  of  perhaps  350.  It  is  dependent  almost 
entirely  upon  the  diet,  being  low  on  a  vegetable  (base-forming  diet) 
and  high  on  a  diet  containing  much  meat,  rice,  whole  wheat  products, 
fruits  containing  benzoic  acid,  as  prunes  and  cranberries,  etc.  (acid- 
forming  foods).  On  the  administration  of  15  grams  of  sodium  bicar- 
bonate it  may  go  down  to  100;  the  ingestion  of  much  acid-forming 
food  may  increase  it  to  600.  In  fasting  it  may  rise  in  a  few  days  to 
800.  It  must  be  borne  in  hiind  that  acidities  of  less  than  250  usually 
indicate  a  true  alkalinity  of  the  urine  inasmuch  as  phenolphthalein 
changes  in  an  alkaline  solution.  Samples  of  urine  collected  shortly 
after  a  meal  may  be  alkaline  due  to  the  so-called  ''alkaline  tide." 

Bacterial  decomposition  of  the  urea  of  the  urine  occurring  in  the 
urinary  tract  will  increase  the  amount  of  ammonia  and  decrease  the 
acidity  of  the  urine.  The  same  change  usually  occurs  in  urine  left  in 
contact  with  the  air.  The  acidity  of  the  urine  is  increased  in  acidosis, 
cardio-renal  and  certain  other  disorders.  The  acidity  of  the  urine 
may  be  somewhat  increased  by  administration  of  mineral  acids,  acid 
phosphates,  or  benzoates,  but  it  is  much  more  difficult  to  increase  than 
to  decrease  this  acidity. 

Hydrogen  Ion  Concentration  or  True  Acidity 

Indicator  Method  (Henderson  and  Palmer's  Adaptation  of  Soren- 
sen's  Method). ^^ — Principle. — The  reaction  of  the  urine  is  estimated 

'  Henderson  and  Palmer:  Tour.  Biol.  Chent.,  13,  393,  1913. 


URINE  481 

by  matching  the  colors  produced  when  a  few  drops  of  indicator  are  added 
respectively  to  the  diluted  urine  and  to  standard  solutions  of  known 
reaction  similarly  diluted.  Similar  hydrogen  ion  concentrations 
are  indicated  by  similar  colors.  The  indicator  must  be  properly 
chosen. 

Standard  Solutions. — A  series  of  standard  solutions  of  known  hy- 
drogen ion  concentration  must  be  prepared.  The  solutions  as  indi- 
cated in  Table  I  (page  482)  are  satisfactory  for  urine  analysis.  The 
table  also  indicates  the  H  ion  concentration  of  each  solution,  the  figure 
given  being  the  logarithm  of  this  concentration  (Ph+).  It  is  more 
convenient  and  rational  to  express  the  concentration  by  this  logarithmic 
notation.  True  H  ion  concentrations  corresponding  to  the  logarithmic 
figures  are  given  in  Table  II  (page  482). 

The  13  solutions  indicated  are  made  up  by  mixing  equal  volumes 
of  their  ingredient  solutions  of  the  composition  indicated.  Solutions 
4  to  12  are  all  that  are  ordinarily  required  as  the  normal  urinary  H 
ion  concentrations  lie  between  4.80  and  7.50  and  pathological  variations 
are  usually  within  these  limits.  The  mean  normal  value  is  almost 
exactly  6.00. 

Procedure. — Select  eleven  250  c.c.  flasks  of  good  glass  and  indistinguishable 
in  color  and  form.  Into  each  of  ten  of  these  introduce  10  c.c.  of  the  various  stand- 
ard solutions.  Make  up  to  250  c.c.  with  distilled  water  and  add  to  each  exactly  the 
same  amount  of  an  aqueous  solution  of  sodiimi  alizarine  sulphonate  (10-15  drops). 
Mix  well  by  inverting.  Introduce  10  c.c.  of  the  urine  to  be  tested  into  a  similar 
250  c.c,  flask,  dilute  and  add  indicator  in  exactly  the  same  way  as  before.  Match 
the  color  of  the  diluted  urine  solution  with  one  of  the  standard  solutions.  By 
consulting  Table  11  (page  482)  determine  to  what  H  ion  concentration  this  corre- 
sponds. This  table  points  out  the  indicators  to  be  used  for  different  ranges  of 
acidity.  From  5.3-6.7  /j-nitrophenol  is  satisfactory  and  is  used  in  the  same  way 
as  aUzarin  except  that  it  must  be  present  in  concentration  of  0.08  per  cent. 
Neutral  red  is  used  in  the  same  way  for  acidities  from  6.7-7.5  about  1.5  c.c.  of 
the  I  per  cent  solution  being  required.  For  acidities  greater  than  5.5  methyl  red 
is  used  in  the  following  way :  10  c.c.  portions  of  the  standard  solutions  are  intro- 
duced into  carefully  selected  colorless  test-tubes  and  10  c.c.  of  urine  is  introduced 
into  another  tube.  The  standard  solutions  are  then  colored  to  match  the  urine 
by  the  addition  of  small  amounts  of  />-nitrophenol,  methyl  orange,  aUzarine  or 
bismark  brown.  Then  to  standard  solutions  and  urine  add  0.15  c.c.  of  a  satu- 
rated solution  in  50  per  cent  alcohol,  of  methyl  red  and  match  the  colors.  For 
concentrations  of  7.5-9.27  or  less  undiluted  urine  is  matched  in  test-tubes  against 
undiluted  standard  solutions,  using  phenolphthalein  as  an  indicator  (without 
previous  coloration  of  standard  solution  >.  In  all  cases  estimations  are  made  in 
duplicate. 


31 


482 


PHYSIOLOGICAL    CHEMISTRY 


TABLE  I 

S'o. 

XaH^PO, 

Xa2HP04 

^H  + 

Indicate 

r 

I 

o.iooo  X 

9.27  " 

2 

o.oooi  X 

0 . 0480  N 

8.7 

Phenolphthalein 

3 

o.oooi  X 

0.0120  X 

8.0 

] 

4 

5 

0.0166  X 
o.ooio  X 

0.0833  ^ 
0 . 0060  N 

7.48 
7.38 

•  Xeutral  red        \ 

6 

o.ooio  X 
CH3COOH 

0.0023  N 
CH3COOX 

6.90 

a 

' 

7 

0.0009  N 

0.0920  X 

6.70 

Sodium  alizarine 

8 

0.0023  X 

0.0920  X 

6.30 

sujphonate. 

9 

0 . 0046  X 

0.0920  X 

6.00 

10 

0.0092  X 

0.0920  X 

S-70 

^Xitrophenol 

II 

0.0230  X 

0.0920  X 

530 

]         ,    ,       , 

12 

0 . 0460  X 

0.0920  X 

4.90 

\  Methyl  red 

13 

0.0920  X 

0.0920  X 

4-70. 

TABLE  II 

Log 

+ 

H 

Log 

+ 
H 

4.6 

25oXio~' 

6.4 

4.0    XlO-7 

4.8 

i6oXio~^ 

6.6 

2.S     XlO-7 

5-0 

looXio"^ 

6.8 

1.6  Xio-^ 

5-2 

63X10-7 

7.0 

i.o  Xio-^ 

5-4 

40X10  7 

7.2 

0.63x10-7 

5-6 

25X10-7 

7-4 

0.40x10-7 

5-8 

16 X 10-7 

7.6 

0.25x10-7 

6.0 

10X10-' 

7.8 

O.I6XIO-7 

6.2 

6.3X10-7 

8.0 

O.IoXlO-7 

Inter pr elation. — The  H  ion  concentration  of  the  urine  is  influenced 
by  the  same  factors  as  the  titratable  acidity  (^see  page  480).  The 
normal  values  lie  between  4.80  and  7.50  with  a  mean  value  of  almost 
exactly  6.00.  For  vegetarians  the  mean  value  is  about  6.64.  In 
cardio-renal  disorders  the  mean  is  5.3.  In  most  pathological  conditions 
the  hvdrogen  ion  concentration  is  increased. 


Total  Solids 

1.  Drying  Method. — Place  5  c.c.  of  urine  in  a  weighed  shallow  dish,  acidify 
very  slightly  with  acetic  acid  (1-3  drops),  and  dry  it  in  vacuo  in  the  presence  of  sul- 
phuric acid  to  constant  weight.  Calculate  the  percentage  of  solids  in  the  urine 
sample  and  the  total  solids  for  the  24-hour  period. 

Interpretation. — The  average  excretion  of  total  solids  by  a  normal  adult  man  is 
about  70  grams.  It  is  largely  dependent  upon  the  protein  and  salts  of  the  diet. 
It  may  be  decreased  in  severe  nephritis  due  to  impaired  excretion,  and  greatly  in- 
creased in  diabetes  with  high  sugar  elimination. 

Practically  all  the  methods  the  technic  of  which  includes  evaporation  at  an 
increased  temperature,  either  under  atmospheric  conditions  or  in  vacuo,  are  attended 
with  error. 

Shackell's  method*  which  entails  the  vacuum  desiccation  of  the  frozen  sample 
is  extremely  satisfactory  and  should  be  used  in  all  biological  work  where  the  great- 
est accuracy  is  desired. 

1  Shackell:  American  Journal  of  Physiology,  24,  325,  1909 


URINE  483 

2.  Calcxilation  by  Long's  Coefficient. — The  quantity  of  solid  material  contained 
in  the  urine  excreted  for  any  24-hour  period  may  be  approximately  computed  by 
multiplying  the  second  and  third  decimal  figures  of  the  specific  gravity  by  2.6. 
This  gives  us  the  number  of  grams  of  solid  matter  in  i  liter  of  urine.  From  this  value 
the  total  solids  for  the  24-hour  period  may  easily  be  determined. 

Calculation. — If  the  volume  of  urine  for  the  24  hours  was  11 20  c.c.  and  the  spe- 
cific gravity  1.018,  the  calculation  would  be  as  follows: 

(c)  18X2.6  =  46.8  grams  of  solid  matter  in  i  liter  of  urine. 

...     46.8X1I2O  r       i-j  ^^        •  t        • 

(0)  -    =52.4  grams  of  solid  matter  m  1120  c.c.  of  unne. 

Long's  coefiicient  was  determined  for  urine  whose  specific  gravity  was  taken 
at  25°C.  and  is  probably  more  accurate,  for  conditions  obtaining  in  America,  than 
the  older  coefficient  of  Haeser,  2.33. 

Interpretation. — See  above. 

Total  Nitrogen 

I.  Kjeldahl  Method  J — Principle. — The  principle  of  this  method  is 
the  conversion  of  the  various  nitrogenous  bodies  of  the  urine  into  am- 
monium sulphate  by  boiling  with  concentrated  sul- 
phuric acid,  the  subsequent  decomposition  of  the 
ammonium  sulphate  by  means  of  a  fixed  alkah 
(NaOH)  and  the  collection  of  the  liberated  ammonia 
in  an  acid  of  known  strength.  Finally,  this  partly 
neutralized  acid  solution  is  titrated  with  an  alkali 
of  known  strength  and  the  nitrogen  content  of  the 
urine  under  examination  computed. 


Procedure. — Place  5  c.c.  of  urine  in  a  500  c.c.  long- 
necked  Jena  glass  Kjeldahl  flask,  add  20  c.c.  of  concen-  Fume^  Absorber^ 
trated  sulphuric  acid  and  about  0.2  gram  of  copper  sulphate 
and  boil  the  mixture  for  some  time  after  it  is  colorless  (about;  one  hour).  If 
a  suitable  hood  or  fimie  chamber  is  not  available  the  sulphuric  acid  vapors  may 
be  carried  away  by  suction.  Coimect  the  outlet  tube  of  a  2-3  liter  wash  bottle 
filled  with  caustic  soda  solution  with  a  suction  pump.  The  inlet  tube  is  connected 
with  a  Folin  fume  absorption  tube  such  as  illustrated  in  Fig.  152.  If  such  a  tube 
is  not  at  hand  a  small  funnel  may  be  attached.  The  absorption  tube  is  placed 
loosely  over  the  mouth  of  the  digestion  flask  and  a  constant  current  of  air  drawn 
through  the  apparatus. 

Allow  the  flask  to  cool  and  dilute  the  contents  with  about  200  c.c.  of  am- 
monia-free water.  Add  a  little  more  of  a  concentrated  solution  of  NaOH  than  is 
necessary  to  neutralize  the  sulphuric  acid-  and  introduce  into  the  flask  a  little 
coarse  pumice  stone  or  a  few  pieces  of  granulated  zinc,^  to  prevent  bumping,  and  a 

'  There  are  numerous  modifications  of  the  original  Kjeldahl  method;  the  one  described 
here,  however,  has  given  excellent  satisfaction  and  is  recommended  for  the  determination  of 
the  nitrogen  content  of  urine. 

*This  concentrated  sodium  hydroxide  solution  should  be  prepared  in  quantity  and 
"check"  tests  made  to  determine  the  volume  of  the  solution  necessary  to  neutralize  the 
volume  (20  c.c.)  of  concentrated  sulphuric  acid  used. 

*  Powdered  zinc  may  be  substituted. 


484  PHYSIOLOGICAL   CHEMISTRY 

small  piece  of  paraffin  to  lessen  the  tendency  to  froth.  By  means  of  a  safety- 
tube  connect  the  flask  with  a  condenser  so  arranged  that  the  delivery-tube  passes 
into  a  vessel  containing  a  known  volume  (the  volume  used  depending  upon  the 
nitrogen  content  of  the  urine)  of  N/io  sulphuric  acid,  using  care  that  the  end  of 
the  delivery-tube  reaches  beneath  the  surface  of  the  fluid.  ^  Mix  the  contents 
of  the  distillation  flask  very  thoroughly  by  shaking  and  distil  the  mixture  imtil 
its  volvune  has  diminished  about  one-half.  Titrate  the  partly  neutralized  N/io 
sulphuric  acid  solution  by  means  of  N/io  sodivmi  hydroxide,  using  congo  red  as 
indicator,  and  calculate  the  content  of  nitrogen  of  the  urine  examined. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  N/io  sodium 
hydroxide  used  in  the  titration  from  the  number  of  cubic  centimeters  of  N/io 
sulphuric  acid  taken.  The  remainder  is  equivalent  to  the  number  of  cubic  centi- 
meters of  N/io  sulphuric  acid,  neutralized  by  the  ammonia  of  the  urine.  One 
c.c.  of  N/io  sulphuric  acid  is  equivalent  to  0.0014  gram  of  nitrogen.  Therefore, 
if  y  represents  the  volimie  of  urine  used  in  the  determination,  and  y'  the  number 
of  cubic  centimeters  of  N/io  sulphuric  acid  neutralized  by  the  ammonia  of  the 
urine,  we  have  the  following  proportion: 

y:  100:: y'Xo. 0014: X  (percentage  of  nitrogen  in  the  urine  examined). 

Calculate  the  quantity  of  nitrogen  in  the  24-hour  urine  specimen. 

Interpretation. — An  adult  of  medium  size  on  a  mixed  diet  will  usually 
excrete  12-18  grams  of  nitrogen  per  day.  It  varies,  hov^^ever,  almost 
directly  with  the  protein  ingestion  and  hence  usually  runs  parallel  to 
the  excretion  of  urea  (see  page  493).  In  a  normal  adult  the  total 
nitrogen  of  the  feces  and  of  the  urine  will  often  be  almost  exactly 
equal  to  the  total  nitrogen  of  the  food.  Such  a  condition  is  called 
''nitrogen  equilibrium."  The  feces  usually  contain  very  little  nitrogen. 
(See  also  Ammonia,  Creatinine,  etc.) 

Calculation  of  Percentage  Nitrogen  Distribution.— In  modern  metabo- 
lism studies  where  the  various  forms  of  nitrogen  are  determined,  in 
addition  to  the  total  nitrogen  as  yielded  by  the  Kjeldahl  method,  it  is 
customary  to  indicate  what  portion  of  the  total  nitrogen  was  present  in 
the  form  of  each  of  the  individual  nitrogenous  constituents.  These 
percentage  values  are  secured  by  dividing  the  weight  (grams)  of 
nitrogen  excreted  for  the  day  in  the  form  of  each  individual  nitrogenous 
constituent  by  the  weight  of  the  total  nitrogen  output  for  the  same 
period.  For  example,  if  the  total  nitrogen  excretion  is  9.814  grams 
and  the  excretion  of  urea-nitrogen  is  8.520  grams  and  the  excretions 
of  nitrogen  in  the  forms  of  ammonia  and  creatinine  are  0.271  gram  and 
0.639  gram  respectively,  the  percentage  distribution  for  these  forms  of 
nitrogen  would  be  calculated  as  follows: 

8.520  grams  urea-nitrogen  -i-  9.814  grams  total  nitrogen   =   84.3  per  cent 

o.  271  gram  ammonia-nitrogen     -r-  9.814  grams  total  nitrogen   =      2.7  per  cent 
0.639  gram  creatinine-nitrogen   "^  9.814  grams  total  nitrogen   =     6.5  per  cent 

^This  delivery-tube  should  be  of  large  caliber  in  order  to  avoid  the  "sucking  back" 
of  the  fluid. 


URINE  485 

Nitrogen  Partition  in  Urines  Containing  Albumin. — If  the  urine  to 
be  tested  contains  albumin  this  must  be  removed  before  an  attempt  at 
a  nitrogen  partition  is  made.  This  may  be  done  by  heating  to  boil- 
ing, acidifying  with  acetic  acid  to  coagulate  the  protein,  filtering  and 
making  up  the  filtrate  to  the  original  volume  of  the  urine.  If  very  small 
amounts  of  albumin  are  present  this  is  attended  with  difficulty.  In 
these  cases  Tracy  and  Welker^  have  suggested  the  use  of  aluminium 
hydroxide  cream.  It  apprently  removes  none  of  the  nitrogenous  con- 
stituents of  normal  urine. 

Procedure. — One  liter  of  urine  (containing  not  over  i  per  cent  of  albvunin) 
is  mixed  with  one  liter  of  aluminium  cream-  and  filtered. 

2.  Folin-Farmer  Microchemical  Method.'' — Principle. — This 
method  belongs  with  the  so-called  microchemical  methods  inasmuch  as 
it  is  adapted  to  the  determination  of  amounts  of  nitrogen  in  the  neigh- 
borhood of  I  mg.  while  in  the  ordinary  Kjeldahl  procedure  30-100  mg. 
of  nitrogen  are  generally  manipulated.  One  c.c.  of  diluted  urine  is 
decomposed  with  sulphuric  acid  as  in  the  Kjeldahl  method,  the  am- 
monia formed  is  set  free  by  the  addition  of  alkali  and  carried  over  into 
an  acid  solution  by  means  of  a  current  of  air.  The  ammonia  solution 
is  then  treated  with  the  Nessler- Winkler  reagent  and  the  color  produced 
compared  with  that  of  a  standard  solution  of  an  ammonium  salt 
treated  in  the  same  way. 

Colorimeter. — For  this  method  as  well  as  for  a  number  of  other 
methods  commonly  used  in  urinary  and  blood  analysis  an  instrument 
known  as  a  colorimeter  is  required.  Through  its  aid  we  are  able  ac- 
curately to  measure  the  respective  depths  of  color  in  two  solutions  and 
hence  to  calculate  the  comparative  amounts  of  substances  which  form 
colored  compounds  in  a  quantitative  manner.  The  most  satisfactory 
instrument  for  this  purpose  is  the  Duboscq  colorimeter  (see  Fig.  153, 
page  486).  This  enables  the  two  colored  solutions  to  be  compared  in 
the  same  optical  field  and  with  a  degree  of  accuracy  of  about  i  per 
cent.  The  later  type  of  the  Duboscq  colorimeter  with  cylinders  instead 
of  prisms  movable  is  to  be  preferred,  particularly  as  this  type  may  be 
readily  adapted  to  the  comparison  of  cloudy  solutions  or  suspensions, 
the  instrument  thus  modified  being  called  a  nephelometer  (see  Fig.  83, 
page  291).     In  this  later  form  of  colorimeter  the  depths  of  the  colored 

1  Tracy  and  Welker:  Jour.  Biol.  Clicni.  22,  55,  1915;  For  other  applications  of  alu- 
minium hydroxide  precipitation  of  colloids,  see  Welker  and  Marshall,  /.  Avi.  Client.  Soc, 
25,  820,    1Q13. 

'^Aluminium  Hydroxide  Cream. — To  a  i  per  cent  solution  of  ammonium  alum  at  room 
temperature  add  a  slight  excess  of  a  i  per  cent  solution  of  ammonium  hydroxide.  Wash  by 
decantation  until  the  wash  water  shows  only  the  faintest  trace  of  residue  on  evaporation. 
Stronger  solutions  should  not  be  used. 

^  Folin  and  Farmer:  Jour.  Biol.  Clirm.,  11,  493,  1912. 


486 


PHYSIOLOGICAL  CHEMISTRY 


solutions  through  which  the  light  passes  are  regulated  by  raising  or 
lowering  the  cups  and  are  accurately  indicated  in  millimeters  on  a 
vernier  scale  at  the  back  of  the  instrument.  The  standard  solution  is 
placed  at  any  convenient  depth  and  the  color  of  the  solution  to  be  ex- 
amined is  matched  with  it  by  raising  or  lowering  cups.  When  the  color 
is  of  the  same  intensity  as  the  standard  the  depth  of  the  solution  is 
read.  The  amounts  of  the  colored  substance  in  solution  are  inversely 
proportional  to  the  depths  of  the  columns  of  fluid.  Thus  if  the  standard 
is  set  at  lo  mm.  and  the  solution  under  examination  has  the  same  color 


Fig.  153. — DuBOSCQ  Colorimeter. 

density  at  20  mm.  the  latter  has  just  one-half  the  concentration  of  the 
standard. 

A  large  number  of  other  colorimeters  have  been  devised  and 
may  be  used  in  place  of  the  Duboscq.  Most  of  these  though  less  ex- 
pensive than  this  instrument  are  also  less  accurate.  The  HelHge 
colorimeter  has  been  recommended,  particularly  for  cHnical  de- 
terminations by  Myers  and  Fine.^  Kober  has  devised  a  combined 
colorimeter  and  nephelometer  which  may  be  obtained  in  this  country 
(see  page  293).     For  merely  approximate  determinations  the  color 

1  Myers  and  Fine:  Post-graduate,  1914-1915. 


URINE 


487 


comparisons  may  be  made  directly  with  a  series  of  colored  standards  of 
varying  strengths  made  up  in  exactly  similar  test-tubes  or  small  flasks. 

Procedure. — Introduce  5  c.c.  of  urine  into  a  50  c.c.  volumetric  fiask  if  the 
specific  gravity  of  the  urine  is  over  10 18,  or  into  a  25  c.c.  fiask  if  the  specific 
gravity  is  less  than  1018.^  Fill  the  fiask  to  the  mark  with  distilled  water  and 
invert  it  several  times  in  order  to  guarantee  thorough  mixing.  Transfer  i  c.c.^ 
of  the  diluted  urine  to  a  large  (20-25  °^™-  X  200  mm.)  Jena-glass  test-tube. 
Add  to  this  i  c.c.  of  concentrated  sulphuric  acid,  i  gram  of  potassium  sulphate, 
I  drop  of  5  per  cent  copper  sulphate  solution  and  a  small,  clean,  quartz  pebble  or 


Q    e 


Fig.  154.  Fig.  155. 

Figs.  154  and  155. — Forms  of  App.\ratus  used  in  Methods  of  Folin  and  Associ- 
ates FOR  Determination  of  Total  Nitrogen,  Urea  and  Ammonia.     (From  Jour.  Biol. 
1912.) 


Chem.   vol. 


glass  bead.  (The  pebble  or  bead  is  added  to  prevent  bumping.)  Boil  the  mix- 
ture over  a  micro-burner'  for  about  six  minutes,  i.e.,  about  two  minutes  after  the 
mixture  has  become  colorless.  Allow  to  cool  until  the  digestion  mixtiu-e  begins 
to  become  viscous.  This  ordinarily  takes  about  three  minutes,  but  in  any  event 
the  mixture  must  not  be  permitted  to  soUdify.  Add  about  6  c.c.  of  water  (a 
few  drops  at  a  time,  at  first,  then  more  rapidly)  to  prevent  sohdification.  To  this 
acid  solution  add  an  excess  of  sodiiun  hydroxide  (3  c.c.  of  a  saturated  solution  is 
sufficient)  and  aspirate  the  Uberated  ammonia  by  means  of  a  rapid  air  current* 

*  The  purpose  is  to  so  dilute  the  urine  that  i  c.c.  of  the  diluted  fluid  shall  contain  0.75- 
1.5  mg.  of  nitrogen. 

^This  measurement  should  be  made  by  means  of  a  modified  Ostwald  pipette  (see  Ost- 
wald-Lulher:  Physiko-Chemische  Messungen,  2d.  ed.,  p.  135).  Such  pipettes  may  be 
obtained  from  Eimer  and  Amend,  New  York. 

'  A  type  of  burner  which  has  proven  satisfactory  is  Eimer  and  Amcnd's  No.  2587. 

*  Either  a  vacuum  pump  or  compressed  air  or  a  force  pump  may  be  used.     The  com- 

{)ressed  air  method  is  rather  the  more  convenient  inasmuch  as  the  ammonia  may  be  col- 
ected  directly  in  a  volumetric  flask.     Inasmuch  as  the  necks  of  such  flasks  (100  c.c.)  are 


488  PHYSIOLOGICAL   CHEMISTRY 

into  a  volumetric  flask  (loo  c.c.)  containing  about  20  c.c.  of  ammonia-free  water 
and  2  c.c.  of  N/io  hydrochloric  acid  (see  Figs.  154  and  155,  page  487).  The 
air  current  should  be  only  moderately  rapid  for  the  first  two  minutes  but  at  the 
end  of  this  two-minute  period  the  current  should  be  run  at  its  maximum  speed 
for  an  interval  of  eight  minutes. 

Disconnect  the  flask,  dilute  the  contents  to  about  60  c.c.  with  ammonia- 
free  water  and  dilute  similarly  i  mg.  of  nitrogen  in  the  form  of  ammonitrai  sul- 
phate^ in  a  second  volimietric  flask.  Nesslerize  both  solutions  as  nearly  as 
possible  at  the  same  time  with  5  c.c.  of  Nessler-Winkler  solution-  diluted,  imme- 
diately before  using,  with  about  25  c.c.  of  ammonia-free  water  to  avoid  turbidity. 
Immediately  fill  the  two  flasks  to  the  mark  with  ammonia-free  water,  mix  well 
and  determine  the  relative  intensity  of  the  two  colors  by  means  of  a  Duboscq 
colorimeter.  3 

The  color  of  the  unknown  should  be  adjusted  to  that  of  the  standard  both  from 
above  and  below  the  level  of  the  latter.  The  matching  of  the  colors  is  ordinarily 
very  easy.  It  is  desirable  to  make  the  readings  by  diffused  dayUght  if  possible. 
If  e'ectric  light  must  be  used,  a  sheet  of  smooth  white  paper  should  be  interposed 
between  the  colorimeter  and  the  source  of  Ught. 

Calculation. — The  reading  of  the  standard  divided  by  the  reading  of  the  un- 
known gives  the  nitrogen  in  milligrams  in  the  volume  of  the  urine  taken.  Calcu- 
late the  total  nitrogen  output  for  the  24-hour  period. 

Interpretation. — See  page  484. 

3.  Bock  and  Benedict's  Modification  of  the  FoUn-Farmer  Procedure. — 
Bock  and  Benedict*  have  found  distillation  of  the  ammonia  more  acciu-ate  than 

not  large  enough  to  permit  of  the  use  of  a  two-hole  rubber  stopper  when  suction  is  used, 
the  ammonia  should  be  collected  in  one  of  the  Jena  test-tubes  previously  described  which 
contains  2  c.c.  of  N/io  hydrochloric  acid  and  about  5  c.c.  of  ammonia-free  water.  The  am- 
monium salt  is  then  transferred  to  the  volumetric  flask  with  40-50  c.c.  of  water  and  Nes- 
slerized  as  described. 

^  Care  should  be  taken  to  secure  the  pure  salt.  All  ammonium  salts  contain  pyridine 
bases  which  titrate  like  ammonia  but  do  not  react  with  Nessler's  reagent.  Pure  ammonium 
sulphate  may  be  prepared  by  decomposing  a  high-grade  ammonium  salt  with  sodium  hy- 
droxide and  passing  the  liberated  ammonia  into  pure  sulphuric  acid.  The  salt  is  then  pre- 
cipitated by  means  of  alcohol,  then  brought  into  solution  in  water  and  re-precipitated  by 
alcohol.  The  final  product  should  be  dried  in  a  desiccator  over  sulphuric  acid.  Dr.  H.  L. 
Emerson  of  Boston  prepares  a  salt  which  is  very  satisfactory  for  use  in  this  method. 
According  to  Bock  and  Benedict,  Kahlbaum's  "Zur  Analyse"  ammonium  chloride  is 
satisfactory. 

^  Chetn.  Zeil.,  1899,  p.  541.     The  Nessler-Winkler  solution  has  the  following  formula: 

Mercuric  iodide 10  grams. 

Potassium  iodide 5  grams. 

Sodium  hydro.xide 20  grams. 

Water 100  c.c. 

The  mercuric  iodide  is  rubbed  up  in  a  small  porcelain  mortar  with  water,  then  washed 
into  a  flask  and  the  potassium  iodide  added.  The  sodium  hydroxide  is  dissolved  in  the 
remaining  water  and  the  cooled  solution  added  to  the  above  mixture.  The  solution  cleared 
by  standing  is  preserved  in  a  dark  bottle. 

The  25  c.c.  portion  of  the  diluted  reagent  should  be  added  about  one-third  at  a  time  to 
the  contents  of  the  flask.  It  is  verj'  essential  that  the  dilution  of  the  reagent  takes  place 
immediately  preceding  its  use,  inasmuch  as  the  diluted  reagent  deteriorates  in  a  few  minutes  as 
is  indicated  by  the  formation  of  a  brick-red  precipitate.  Fortunately  the  reagent  does  not 
decompose  in  this  manner  in  the  presence  of  the  ammonium  salt. 

^  The  standard  may  be  set  at  any  desired  depth  but  a  very  satisfactory  depth  is  20  mm. 
The  depth  should  be  uniform  throughout  any  series  of  comparative  tests. 

*Bock  and  Benedict:  Jour.  Biol.  Cliem.,  20,  47,  1915. 


URINE 


489 


aspiration.  They  connect  the  large  Jena  test-tube  in  which  the  digestion  was 
carried  out  with  a  small  Liebig  condenser  (made  from  a  piece  of  glass  tubing 
30  by  150  mm.  with  two-hole  rubber  stoppers  at  each  end  through  which  pass 
the  inlet  and  outlet  tubes  and  the  condenser  tube  itself  j.  See  Fig.  156.  The  lower 
end  of  the  condenser  is  connected  with  a  glass  tube  (or  better  an  old  pipette,  to 
prevent  back  suction)  which  reaches  nearly  to  the  bottom  of  the  voliunetric  flask 
used  as  a  receiver.  The  distillation  tube  also  has  a  two-hole  rubber  stopper.  It 
is  connected  with  the  condenser  and  also  carries  a  long  straight  tube  which  reaches 
nearly  to  the  bottom  of  the  test-tube,  and  is  closed  above  with  a  piece  of  rubber 
tubing  and  a  pinch-cock.  The  digestion  is  carried  out  just  as  in  the  Folin-Farmer 
method  (see  page  485)  and  when  partially  cool  7  c.c.  of  water  are  added.  Into  the 
long  tube  passing  through  the  stopper  suck  3  c.c.  of  saturated  sodium  hydroxide 
solution  and  close  the  pinch-cock.     Insert  the  stopper,  connect  with  the  condenser 


Fig.  156. — Bock  and  Benedict  Apparatus. 

and  allow  the  alkah  to  run  into  the  test-tube.  The  fluids  are  mixed  by  blowing  a 
few  bubbles  of  air  through  the  apparatus.  The  test-tube  is  then  heated  to  vigor- 
ous boiling  (over  a  large  free  flame),  the  distillation  being  continued  until  a  sepa- 
ration of  salts  occurs  in  the  test-tube  and  the  mixture  begins  to  bump.  This 
distillation  requires  about  two  minutes.  The  test-tube  is  then  disconnected  from 
the  condenser  and  the  latter  washed  down  with  a  few  cubic  centimeters  of  water. 
The  liquid  in  the  receiving  flask  is  diluted  and  Nesslerized  as  in  the  Folin-Farmer 
method  (see  page  485). 

Bock  and  Benedict,  while  holding  the  distillation  procedure  to  be 
more  accurate  than  aspiration,  do  not  consider  that  the  colorimetric 
method  is  equivalent  to  the  standard  Kjeldahl  procedure  in  accuracy 
or  reliability,  although  usually  it  agrees  with  the  latter  method  within 
about  2-3  per  cent,  and  is  indispensable  where  very  small  amounts  of 
nitrogen  are  to  be  determined.  According  to  Folin^  and  others  the 
method  is  capable  of  greater  accuracy  than  this,  and  the  aspiration 

*  Folin:  Jour.  Biol.  Cfictn.,  21,  195,  1915. 


490  PHYSIOLOGICAL   CHEMISTRY 

procedure  gives  satisfactory  results.  The  method  should  be  checked 
up  carefully  by  each  new  learner  of  the  method,  using  pure  solutions. 
Outside  air  is  better  than  laboratory  air  for  aspiration  purposes. 
Care  is  needed  in  using  the  pipettes,  which  should  be  of  the  Ostwald 
type  and  accurate.  In  using  them  allow  the  pipette  to  drain  against 
the  side  of  the  vessel  for  lo  seconds  and  then  blow  out  clean  so  that 
nothing  is  left  behind  in  the  tip.  The  reagents  used  must  be  as  free 
as  possible  from  ammonia  and  must  be  checked  up,  particularly  the 
sulphuric  acid  and  potassium  sulphate.  Those  who  have  trouble  in 
using  a  colorimeter  may  substitute  titration  with  N/50  hydrochloric 
acid  using  alizarin,  or  better  methyl  red,  as  an  indicator. 

4.  Gulick's  Modification  of  the  Folin -Fanner  Colorimetric  Method.' — Prin- 
ciple.— By  using  small  amounts  of  sulphuric  acid  and  potassium  sulphate  it  is  possi- 
ble to  Nesslerize  the  products  of  the  digestion  directly 
without    the    necessity    of    previous    aspiration   of    the 
ammonia. 

Procedure. — Dilute  the  sample  of  urine  to  an  exact 
multiple  of  its  original  volume  (about  4-10  times)  with 
acid  mixture,^  so  that  0.5  c.c.  of  the  dilution  will  contain 
between  0.4  and  0.7  mg.  of  nitrogen.  With  an  Ostwald 
pipette  introduce  0.5  c.c.  of  this  mixture  into  the  bulb 
of  a  micro-oxidation  flask.  The  type  illustrated  in  Fig. 
157,  with  a  bulb  capacity  of  about  15  c.c,  is  the  most 
convenient.  Add  also  a  small  spherical  glass  bead  or 
scrap  of  platinum  or,  better,  a  piece  of  platinum  wire  4-5 
mrn.  long  bent  into  a  tight  spiral.  Agitate  continually 
while  boiling  off  the  water  over  a  micro-burner  (about 
one  minute).  Set  up  to  heat  over  a  very  small  flame  well 
guarded  against  the  wind.  A  luminous  flame  6-7  mm. 
CRO-oxiDATioN  Flask^'  ^^^^  comiug  from  a  burner  tube  of  about  3  mm.  outside 
diameter  may  be  used.  The  digestion  bulb  is  set  at  a 
slant  so  that  the  mouth  is  directed  upward  and  the  heat  applied  at  the  tip. 
Heat  for  at  least  one  minute  after  the  acid  becomes  clear  white.  The  boiling  and 
oxidation  require  about  6-10  minutes.  As  soon  as  the  glass  is  cool  enough  to  bear 
water  add  sufficient  ammonia-free  water  to  dissolve  the  contents,  and  rinse  quanti- 
tatively into  a  50  c.c.  volumetric  flask. 

Introduce  into  a  second  50  c.c.  flask  0.5  mg.  of  nitrogen  in  the  form  of  pure 
ammonium  sulphate.  Fill  both  flasks  to  about  40  c.c.  with  ammonia-free  water. 
Into  each  of  the  flasks  then  inject  5  c.c.  of  the  modified  Winkler  solution^  in  a  vigor- 
ous stream  from  a  pipette.     Fill  to  the  mark  and  mix  thoroughly.     Compare 

^  Gulick:  Jour.  Biol.  Client.,  18,  541,  1914. 

^  Acid  Oxidizing  Mixture. — To  125  c.c.  of  ammonia-free  water  add  40  c.c.  of  sulphuric 
acid,  5  c.c.  of  a  saturated  solution  of  mercuric  chloride  and  20  gm.  of  potassium  sulphate. 
Then  make  up  to  200  c.c.  with  ammonia-free  water. 

^  Modified  Winkler  Solution.— Dissolve  40  grams  of  sodium  hydroxide  in  about  200  c.c. 
of  ammonia-free  water.  Mix  15  grams  of  mercuric  iodide  and  10  grams  of  potassium  iodide 
and  dissolve  in  about  15  c.c.  of  water.  Transfer  with  the  aid  of  the  alkali  to  a  500  c.c. 
volumetric  flask  and  make  up  to  500  c.c.  with  ammonia-free  water.  Transfer  to  an  Erlen- 
meyer  flask  and  let  stand  24  hours  to  settle. 


URINE  491 

at  once  or  at  least  within  an  hour  in  a  colorimeter,  using  a  depth  of  standard  of 
20-30  mm. 

Urea 

I.  Urease  Methods. — Principle. — These  methods  depend  upon  the 
principle  that  the  enzyme  urease  is  able,  at  ordinary  temperatures,  to 
transform  urea,  quickly  and  completely,  into  ammonium  carbonate. 
Takeuchi^  in  1909  discovered  the  presence  of  this  enzyme  in  the  soja 
or  soy  bean.  The  application  of  this  enzyme  to  the  determination 
of  urea  in  urine,  blood,  etc.,  was  first  proposed  by  Marshall,-  whose 
methods  have  been  modified  by  Van  Slyke  and  CuUen.^  These  latter 
investigators  prepared  a  permanent  preparation  of  the  enzyme,  in  a 
water-soluble  form,  the  use  of  which  makes  more  convenient  the  rapid 
and  accurate  determination  of  urea  in  urine,  blood  and  other  biological 
fluids. 

The  urease  method  is  probably  the  most  satisfactory  of  all  methods 
for  the  determination  of  urea.  Other  nitrogenous  constituents  such 
as  allantoin  are  not  decomposed  by  urease.  The  method  involves  no 
carefully  regulated  heating  procedures,  and  is  apphcable  to  diabetic 
urines. 

The  procedure  for  the  determination  in  urine  consists  in  treating 
the  urine  sample  with  urease,  aerating  the  ammonia  formed  into  fiftieth- 
normal  acid,  and  titrating  the  excess  of  acid  with  fiftieth-normal 
alkali.     (For  colorimetric  procedure  see  page  492.) 

Preparation  of  Solid  Urease.* — Digest  one  part  of  soy  bean  meal  with  five  parts 
of  water  at  room  temperature,  with  occasional  stirring,  for  an  hour,  and  clear  the  solu- 
tion by  filtration  through  paper  pulp  or  centrifugation.  Pour  this  extract  slowly, 
with  stirring,  into  at  least  10  volumes  of  acetone.  The  acetone  dehydrates  the 
enzyme  preparation.  Filter,  dry  in  vacuum,  and  powder.  The  activity  of  the 
preparation  is  retained  indefinitely.  Thus  prepared  it  is  not  perfectly  soluble  in 
water,  but  this  fact  interferes  in  no  way  with  its  use. 

Standardization  of  the  Enzyme  Preparation. — Make  up  accurately  a  3  per  cent 
solution  of  pure  urea.  Treat  this  solution  exactly  as  the  urine  is  treated  in  the 
following  method,  using  1.2  c.c.  of  the  solution.  The  ammonia  formed  should 
neutralize  25  c.c.  of  N/50  acid.  If  it  does  so  the  preparation  is  of  sufiicient  strength 
to  use  as  indicated.  If  not,  more  of  the  preparation  must  be  used  for  a 
determination. 

The  ground  soy  bean  may  also  be  used  directly  in  this  determination.  It 
should  pass  through  a  20-mesh  sieve.     Rose  and  Coleman  for  their  micro-procedure 

1  Takeuchi:  7o»r«.  Coll.  Agr.  Tokyo,  1909,  Part  i. 

*  Marshall,  E.  K.,  Jr. :/.  Biol.  CItcm.,  14,  283,  1913;   15,  495,  1913;  15,  487,  1913;  17, 

351,  iQU- 

^Van  Slyke,  D.  D.,  and  Cullen,  G.  E.:  /.  .Iw;.  Med.  Ass'n,  62,  1558,  1914.  See,  also, 
J.  Biol.  CItcm.,  19,  141,  1914. 

*  Van  Slyke  and  Cullen:  Jour.  Biol.  Chem.,  19,  211,  1914.  Satisfactory  preparations 
of  Urease  may  be  obtained  from  the  Arlington  Chemical  Company,  Yonkers,  N.  Y.,  and 
from  Hynson,  Westcott  and  Co.,  Baltimore. 


492 


PHYSIOLOGICAL    CHEMISTRY 


(see  below)  use  0.2-0.4  gram  of  bean  flour  acting  in  a  water-bath  at  50-60°  for  five 
minutes.  In  their  macro-method,  using  5  c.c.  of  urine  they  dilute  with  30  c.c.  of 
water  warmed  to  50-60°  and  then  add  5  grams  of  the  soy  bean  flour  and  let  stand 
for  30  minutes.  They  then  add  5  c.c.  of  saturated  sodium  carbonate  solution  and 
aerate  as  usual. 

(a)  Procedure  of  Van  Slyke  and  Cullen. — Dilute  5  c.c  .of  urine  to  50  c.c.  with 
ammonia-free  water.  Measure  5  c.c.  of  the  diluted  urine  into  Tube  "A"  (see 
Fig.  158),  add  i  drop  of  caprylic  alcohol  (to  prevent  frothing),  and  i  c.c. 
of  enzyme  solution.  ^  Close  "A"  with  stopper  shown  in  figure,  and  let  the  tube 
stand  15  minutes  for  the  enzyme  to  act.  Measure  into  Tube  "B"  25  c.c.  of  N/50 
HCl  or  H2SO4.  Add  i  drop  of  capryUc  alcohol  and  i  drop  of  a  i  per  cent  alizarin 
solution,^  as  indicator.     Connect  "A"  and  "B"  as  shown  in  the  figure.     At  the 

end  of  15  minutes  aspirate  for  about  one-half 
minute  to  remove  any  ammonia  present  in  the 
free  condition  in  "A."  After  this  aspiration, 
open  "A"  and  introduce  5  c.c.  of  saturated 
potassium  carbonate.  Close  "A"  at  once  and 
aspirate  until  all  the  ammonia  has  been  re- 
moved from  "A"  and  carried  over  into  the  acid 
in  "B."  The  time  needed  for  the  aspiration 
varies  for  different  pumps  from  5  to  30  minutes, 
and  should  be  determined  by  trial  for  the  par- 
ticular apparatus  used.  At  the  end  of  the  time 
needed  for  the  aeration,'  the  pvmip  is  discon- 
nected (care  being  taken  to  avoid  back  suc- 
tion) and  the  excess  acid  in  "B"  is  titrated  by 
means  of  fiftieth-normal  alkali. 

Calculations. — The  number  of  cubic  centi- 
meters of  fiftieth-normal  acid  neutralized  is 
multiplied  by  the  factor  0.056  to  give  the 
niunber  of  grams  of  urea-plus  ammonia-nitro- 
gen in  100  c.c.  of  the  urine.  The  ammonia 
alone  may  be  determined  at  the  same  time  as  the  ammonia  plus  urea,  using 
the  same  technic  except  that  5  c.c.  of  the  undiluted  urine,  no  urease,  and  the 
factor  0.0056  are  used  for  the  determination  of  ammonia  alone.  The  am- 
monia tubes  are  run  in  the  same  series  as  those  for  the  urea  determination, 
using  the  same  air  current  for  all. 

(b)  Color imetric  Modification. — Rose  and  Coleman^  suggest  the  colorimetric 
determination  of  the  ammonia  which  is  carried  over  by  the  aspiration,  rather  than 
titration  of  the  excess  of  acid.  They  Nesslerize  the  solution  in  "B,"  and  compare 
the  color  produced  with  the  color  of  a  Nesslerized  solution  of  known  ammonia  con- 
tent, as  in  the  Folin-Farmer  method  for  total  nitrogen.  If  this  procedure  is  fol- 
lowed, the  amount  of  urea  and  ammonia  nitrogen  in  the  solution  acted  upon  by  the 

1  The  enzyme  solution  is  prepared  by  dissolving  2  grams  of  the  enzyme  preparation,  0.6 
gram  of  dipotassium-hydrogen  phosphate,  and  0.4  gram  of  monopotassium-dihydrogen 
phosphate  in  10  c.c.  of  water.  Solution  is  aided  by  stirring  with  a  glass  rod.  The  slightly 
opalescent  solution  should  be  covered  with  toluol  and  may  be  kept  for  two  weeks  without 
losing  activity. 

*  Folin  states  that  methyl  red  is  preferable  to  alizarin  for  ammonia  titrations. 

^See  Fiske  {Jour.  Biol.  Chew.  23,  455,  1915)  and  Van  Slyke  and  Cullen  (Jour.  Biol. 
Chem.,  24,  117,  1916)  for  discussion  of  details  of  method. 

*Rose  and  Coleman;  Biochem.  Bull.,  3,  411,  1914. 


Fig.  158. — Van  Slyke  and 

CtJLLEN  APPAR.A.TUS. 


URINE  493 

urease  must  not  exceed  2  mg.     This  procedure  has  been  found  useful  where  small 
quantities  of  urea  are  to  be  estimated. 

Interpretation. — The  mean  average  daily  excretion  of  urea  by  normal 
adults  is  usually  placed  at  about  30-35  grams  but  is  very  closely  de- 
pendent upon  the  protein  ingestion  and  hence  may  vary  widely.  It 
is  of  significance  only  when  the  amount  of  nitrogen  ingested  is  known 
with  some  degree  of  accuracy.  In  disorders  associated  with  increased 
tissue  catabolism  as  in  fevers,  the  excretion  of  urea  is  increased.  It 
may  be  decreased  in  pronounced  kidney  and  liver  disorders  due  to 
decreased  formation  and  decreased  power  of  elimination,  but  these 
findings  are  not  constant. 

The  per  cent  of  the  total  nitrogen  of  the  urine  occurring  as  urea 
varies  on  the  average  from  80-90.  On  a  high  protein  diet  it  is  nearer 
90  per  cent;  on  a  very  low  nitrogen  but  high  calorie  diet  it  may  not 
be  over  60  per  cent.  In  marked  acidosis  it  may  be  considerably 
decreased  relative  to  the  total  nitrogen  (see  ammonia). 

(c)  Marshall's  Urease  Method.' — Principle. — This  is  a  simple  clin- 
ical method  for  the  determination  of  urea  in  urine.  It  differs  from  the 
preceding  method  in  that  instead  of  aspirating  off  the  ammonia  formed 
from  the  urea  by  the  action  of  the  urease,  it  is  titrated  directly  in  the 
urine  mixture,  thus  simplifying  the  procedure.  The  method  is  nearly 
as  accurate  as  the  preceding,  for  normal  urine  the  error  being  only 
about  2  per  cent  which  is  very  satisfactory  for  a  rapid  clinical  procedure. 
For  diabetic  urines  the  aeration  procedure  should  be  used  as  such  urines 
contain  substances  which  render  the  titration  inaccurate. 

Procedure. — Two  5  c.c.  portions  of  the  urine  are  measured  into  flasks  of 
200-300  c.c.  capacity  and  diluted  with  distilled  water  to  about  100-125  c.c. 
One  c.c.  of  a  10  per  cent  solution  of  urease  prepared  as  described  on  page  491  is 
added  to  one  flask,  a  few  drops  of  toluene  to  each  and  the  solution  allowed  to 
remain,  well  stoppered,  at  room  temperature  over  night  (or  five  hours).  The 
fluid  in  each  fiask  is  titrated  to  a  distinct  pink  color  with  N  10  hydrochloric  acid 
using  methyl  orange  as  an  indicator.  A  few  cubic  centimeters  of  the  enzyme 
solution  used  should  also  be  titrated  to  determine  the  amount  of  N/io  hydro- 
chloric acid  required  to  neutraUze  i  c.c. 

Calculation. — The  amount  of  hydrochloric  acid  required  for  the  contents  of 
the  flask  containing  the  urine  and  enzyme  solution,  less  the  amount  used  for 
5  c.c.  of  urine  alone  and  that  previously  determined  for  i  c.c.  of  enyzme  solution, 
corresponds  to  the  urea  originally  present  in  the  sample  of  urine.  Since  i  c.c. 
of  N/io  HCl  is  equivalent  to  3  mg.  of  urea,  the  number  of  cubic  centimeters 
required,  multiplied  by  0.6  gives  the  value  of  urea  expressed  in  grams  per  Uter 
of  urine. 

Interpretation. — See  above. 

'  Marshall:  Jour.  Biol.  Cliem.,  14,  283,  1913. 


494  PHYSIOLOGICAL   CHEMISTRY 

2.  Benedict's  Method.^ — Principle. — The  urea  is  decomposed  by 
heating  with  a  mixture  of  potassium  bisulphate  and  zinc  sulphate.  The 
fact  that  the  hydrolyzing  agent  is  a  salt  and  that  the  digestion  takes 
place  in  the  practical  absence  of  water  seem  to  insure  less  decomposition 
of  substances  other  than  urea.  The  ammonia  formed  is  distilled  off 
and  determined  in  the  usual  manner. 

Procedure. — Five  c.c.  of  urine  are  introduced  into  a  rather  wide  Jena  glass 
test-tube,  about  3  grams  of  potassium  bisulphate  and  1-2  grams  of  zinc  sulphate* 
added,  a  small  quantity  of  powdered  pumice  and  a  bit  of  parafl&n  are  introduced 
and  the  mixture  boiled  almost  to  drjrness  either  over  a  free  flame  or  by  immersion 
in  a  sulphuric  acid  bath  at  about  130°.  The  tubes  are  then  weighted  (a  screw 
clamp  is  convenient)  and  immersed  for  three-fourths  of  their  length  in  a  bath  of 
sulphuric  acid  at  a  temperature  of  162-165°  (not  lower)  for  one  hour. 

The  contents  of  the  tube  are  then  washed  into  an  800  c.c.  Kjeldahl  distillation 
flask,  diluted  to  about  400  c.c.  with  water,  made  alkaline  by  the  addition  of  15-20 
c.c.  of  10  per  cent  KOH  (or  25  c.c.  15  per  cent  Na2C03)  and  distilled  as  usual  in  the 
Kjeldahl  method  (page  483).  The  value  obtained  must  be  corrected  for  ammonia 
by  a  separate  determination  of  the  latter. 

Welker^  has  suggested  an  electrical  bath  for  use  in  the  first  part  of  this  method. 

3.  Microchemical  Method  of  Folin  and  VeMihone^^Principle. — 

The  urine  is  heated  with  potassium  acetate  and  acetic  acid  to  hydro- 
lyze  the  urea.  The  desired  temperature  is  maintained  with  the  aid 
of  a  temperature  indicator.  The  ammonia  formed  is  aspirated  ofT 
and  determined  colorimetrically  with  Xessler-Winkler  reagent. 

Procedure. — Dilute  the  urine  so  that  1  c.c.  contains  0.75-1.5  mg.  of  urea 
nitrogen.  Generally  dilutions  of  i :  20  or  i :  10,  depending  on  the  concentration, 
are  satisfactory.  By  means  of  an  Ostwald  pipette  (see  page  487)  introduce  i  c.c. 
of  the  diluted  urine  into  a  large  dry  Jena  test-tube  (20-25  mm.  by  200  mm.)  which 
already  contains  7  grams  of  dry  ammonia-free  potassium  acetate^  (free  from 
lumps),  I  c.c.  of  50  per  cent  acetic  acid,  a  small  sand  pebble  or  a  little  powdered  zinc 
(not  zinc  dust)  to  prevent  bumping  during  boiling  and  a  temperature  indicator." 

^  Benedict:  Jour.  Biol.  Chem.,  8,  405,  191 1. 

^  An  excess  of  zinc  salt  is  to  be  avoided  as  too  large  quantity  tends  to  cause  slight  frothing 
during  the  final  distillation. 

'  Welker:  Biochem.  Bull.,  i,  439,  191 2. 

*  Folin  and  Pettibone:  Jour.  Biol.  Chem.,  11,  513,  1912. 

^  A  satisfactory  preparation  containing  less  than  i  per  cent  of  moisture  and  free  from 
ammonia  maj'  be  obtained  from  J.  T.  Baker  Chemical  Co.,  Phillipsburg,  N,  J. 

•  "This  temperature  indicator  consists  of  powdered  chloride-iodide  of  mercury  (HglCl) 
inclosed  in  a  sealed  glass  bulb  not  over  i  mm.  in  diameter.  This  salt  is  bright  red  at  ordi- 
nary temperatures.  At  ii8°C.  it  turns  yellow  and  melts  to  a  clear  dark  red  liquid  at  i55°C. 
It  solidifies  again  at  about  i48°C.  and  resumes  its  red  color  gradually  ordy  in  the  course  of 
about  24  hours.  The  melting-point  temperature,  i53°C.,  is  fortunately  a  temperature 
very  readily  obtained  and  maintained  by  means  of  potassium  acetate  and  as  the  acetate 
begins  to  cake  and  solidify  at  i6o°-i6i°C.,  there  is  no  danger  in  this  combination  of  having 
either  too  high  or  too  low  a  temperature  without  its  being  unmistakably  apparent. 

The  HglCl  may  be  prepared  by  heating,  in  a  dry  state,  intimately  mixed  mercuric 
chloride  and  mercuric  iodide  in  molecular  proportions  at  i5o°-i6o°C.  for  6-8  hours.  At  the 
end  of  the  heating  the  product  should  be  powdered  and  used  as  it  is  for  it  cannot  be  purified 
by  the  use  of  solvents.  It  should  be  kept  dry  until  sealed  up  as  indicated."  These  tern* 
perature  indicators  may  be  obtained  ready  prepared  in  tubes  from  Eimer  and  Amend 
New  York. 


URINE  495 

Close  the  test-tube  by  means  of  a  rubber  stopper  carrying  an  empty  narrow 
"calcium  chloride  tube"  (1.5  cm.  by  25  cm.,  without  bulb)  as  a  condenser.  Sus- 
pend the  test-tube  and  condenser  above  a  micro-burner  (see  page  487  •  by  means 
of  a  burette  clamp  or  some  similar  device  in  such  a  way  that  they  may  be  easily 
raised  or  lowered.  Heat  gently,  using  a  bottomless  beaker  or  some  similar 
device  as  a  wind  shield  if  needed.  The  acetate  will  soon  dissolve  (two  minutes) 
and  the  mixture  begin  to  boil.  At  this  point  the  indicator  begins  to  melt  showing 
that  the  desired  temperature  (i53-i6o°C.)  has  been  reached.  Continue  the 
boiling  in  a  gentle,  even  manner  for  ten  minutes  at  the  end  of  which  time  the 
decomposition  of  the  urea  is  complete.  Remove  the  apparatus  from  the  flame 
and  dilute  the  contents  with  5  c.c.  of  water. ^  Add  an  excess  of  alkali  12  c.c.  of  a 
saturated  solution  of  sodium  hydroxide  or  potassium  carbonate]  and  remove 
the  Uberated  ammonia  by  means  of  a  strong  air  current  (see  page  487).  The 
ammonia  may  be  caught  in  a  100  c.c.  volumetric  flask  which  contains  about  35 
c.c.  of  ammonia-free  water  and  2  c.c.  of  N/io  acid.  With  a  stiong  air  current 
this  process  requires  only  about  ten  minutes.  Determine  the  ammonia  colori- 
metrically  against  i  mg.  of  nitrogen  in  the  form  of  ammonium  sulphate.  For 
the  colorimetric  procedure  see  the  total  nitrogen  determination  page  485. 

4.  Method  of  Folin  and  Denis. ^^ — Principle. — Sugar  interferes  with  the  de- 
composition of  urea.  This  was  formerly  believed  to  be  due  to  the  formation  of 
nitrogenous  "melanins,"*  but  is  more  probably  due  to  the  formation  of  definite, 
stable  ureids.*  This  difficulty  may  be  overcome  by  proper  dilution  of  the  urine 
thus  preventing  the  formation  of  the  ureids.  Because  of  this  great  dilution  titration 
procedures  are  inapplicable,  and  the  colorimetric  procedure  is  applied. 

Procedure. — Dilute  i  c.c.  of  the  urine  with  20  to  100  volumes  of  ammonia-free 
water  and  decompose  i  c.c.  of  this  dilute  urine  with  potassium  acetate  and  acetic 
acid  as  described  under  the  method  of  Folin  and  Pettibone  on  page  494. 

By  means  of  an  air  current  remove  the  ammonia  to  a  second  test-tube  which 
contains  about  2  c.c.  of  water  and  0.5  c.c.  of  N/io  hydrochloric  acid.  Add  to  the 
contents  of  this  tube  about  2  c.c.  of  water  and  3  c.c.  of  the  diluted  (1:5)  Nessler- 
Winkler  solution  (page  490).  Wash  this  colored  solution  into  a  10  c.c.  volumetric 
flask  and  dilute  it  to  the  mark  with  ammonia-free  water.  Transfer  the  entire  vol- 
ume to  a  dry  cylinder  of  a  Duboscq  colorimeter  and  determine  the  depth  of  color 
against  a  standard  containing  i  mg.  of  nitrogen  per  100  c.c.  of  solution.  For  the 
detailed  colorimetric  procedure  see  the  method  for  total  nitrogen,  page  494. 

5.  Method  of  Folin. ^ — Principle. — The  urea  is  decomposed  by  heating  with 
magnesium  chloride  and  hydrochloric  acid.  The  ammonia  is  distilled  off  and  de- 
termined by  titration.  This  method  was  one  of  the  first  accurate  methods  for  the 
determination  of  urea.  .\lIantoin  is  also  included  in  the  results  from  this  method 
but  occurs  only  in  minute  quantities  in  human  urine.  The  method  is  not  applicable 
to  urines  containing  sugar  as  ureides  are  formed.  For  such  urines  the  method  of 
Folin  and  Denis  (above)  or  the  urease  method  using  aspiration  (page  491)  may 
be  employed.     The  earlier  Morner-Sjoqvist  method*  has  been  combined  with  that 

^  This  water  should  be  added  by  means  of  a  pipette  through  the  calcium  chloride  tube  so 
as  to  rinse  the  sides  of  the  tube  and  the  bottom  of  the  rubber  stopper  from  any  possible  traces 
of  ammonium  acetate.     Not  more  than  5  c.c.  of  water  should  be  used  for  this  purpose. 

-Folin  and  Denis:  Jour.  Biol.  Chem.,  11,  520,  1912. 

'Morner:  Skatid.  Arch.  Physiol.,  14,  319. 

*  Folin:  Am.  Jour.  Physiol.,  13,  46,  1905. 

'Folin:  Am.  Jour.  Physiol.,  13,46,  1905. 

•M6rner:  Skand.  Arch.  Physiol,  14,  297,  1903.     See  also  Fourth  Edition  of  this  book. 


496 


PHYSIOLOGICAL   CHEMISTRY 


Q^^mJ* 


of  the  Folin  procedure  in  such  a  way  as  to  render  the  latter  applicable  to  urines 
containing  sugar,  to  remove  certain  interfering  substances  such  as  allantoin  and 
make  the  separate  determination  of  ammonia  unnecessary.  The  method  is,  how- 
ever, time-consuming  and  the  other  methods  mentioned  are  on  this  account  to  be 
preferred  for  the  analysis  of  diabetic  urines. 

Procedure. — Place  5  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer  flask  and  add  to  it 
5  c.c.  of  concentrated  hydrochloric  acid,  20  grams  of  crystallized  magnesium  chlo- 
ride, a  piece  of  paraffin  the  size  of  a  hazel  nut,  and  2-3  drops  of  a  i  per  cent  aqueous 

solution  of  "alizarin  red."  Insert  a  Folin  safety 
tube  (Fig.  159)  into  the  neck  of  the  flask  and 
boil  the  mixture  until  each  drop  of  reflow  from 
the  safety  tube  produces  a  very  perceptible  bump; 
the  heat  is  then  reduced  somewhat  and  con- 
tinued one  and  one-half  hours.  The  contents 
of  the  flask  must  not  remain  alkaline,  and  to 
obviate  this,  at  the  first  appearance  of  a  red- 
dish tinge  in  the  contents  of  the  flask  a  few  drops 
of  the  acid  distillate  are  shaken  back  into  the 
flask.  At  the  end  of  one  and  one-half  hours  the 
contents  of  the  vessel  are  transferred  to  a  i -liter 
flask  with  about  700  c.c.  of  distilled  water,  about 
20  c.c.  of  ID  per  cent  potassium  hydroxide  or  so- 
dium hydroxide  solution  is  added  and  the  mix- 
ture distilled  into  a  known  volume  of  N/io  sul- 
phuric acid  until  the  contents  of  the  flask  are 
nearly  dry  or  until  the  distillate  fails  to  give  an 
alkaline  reaction  to  litmus,  showing  the  absence 
of  ammonia.  The  time  devoted  to  this  process 
is  ordinarily  about  an  hour.  Boil  the  distillate  a 
few  moments  to  free  it  from  CO2,  then  cool  and 
titrate  the  mixture  with  N/io  sodium  hydroxide, 
using  "alizarin  red"  as  indicator. 

A  "  check  "  experiment  should  always  be  made 

to   determine  the  original  ammonia  content  of 

the  urine  and  of  the  magnesium  chloride,  if  it 

is  not  absolutely  pure,  which  of  course  should  be 

subtracted  from  the  total  amount  of  ammonia 

as  determined  by  the  above  process. 

6.  Hypobromite  Methods. — Principle. — The  hypobromite  methods  are  based 

upon  the  fact  that  urea  is  decomposed  by  a  solution  of  sodium  hypobromite  with 

the  formation  of  carbon  dioxide  and  gaseous  nitrogen  according  to  the  following 

equation : 

CO(NH)  2-f  3NaOBr  =  Na-f- CO2+ 2H20-h  3NaBr 


Fig.  i5g. — Folin's  Urea 
App.'Vratus. 


The  carbon  dioxide  is  retained  by  the  alkaline  solution  and  the  nitrogen  gas  is  col- 
lected and  measured.  On  account  of  its  simplicity  this  has  been  the  most  widely 
used  of  clinical  methods  for  the  determination  of  urea.  It  possesses,  however,  sev- 
eral essential  inaccuracies  among  which  may  be  mentioned  the  fact  that  all  of  the 
urea  nitrogen  is  not  liberated,  while  on  the  other  hand  substances  always  present 


URINE 


497 


in  urine  such  as  ammonia,  creatinine,  and  uric  acid  yield  nitrogen  more  or  less 
rapidly  when  the  urine  is  treated  with  hypobromite.  The  measurement  of  the  gas 
as  ordinarily  carried  out  involves  considerable  errors.  The  results  are  usually  high, 
and  may  be  excessively  so.  Robinson  and  Muller^  have  suggested  that  the 
ureometcr  be  shaken  for  five  minutes  to  hasten  the  reaction  and  that  the  results 
as  obtained  be  multiplied  by  the  factor  0.917  to  insure  more  accurate  results. 
They  use  the  Doremus-Hinds  apparatus. 

(a)  Hypobromite  Method  {using  the  Doremus-Hinds  Ureometer). — The  Doremus- 
Hinds  ureometer  (Fig.  160)  is  one  of  the  simplest  and  cheapest  forms  of  appa- 


r^ 


ratus  in  general  use  for  the  determination  of 
urea  by  the  hypobromite  process.  It  is,  how- 
ever, much  less  accurate  than  those  types  of 
apparatus  which  involve  the  measurement  of 
the  nitrogen  in  a  gas  burette  over  water  w'ith 
equalization  of  pressure.  In  using  this  appa- 
ratus proceed  as  follows:  Fill  the  side  tube  B 
and  the  lumen  of  the  stop-cock  C  with  the 
urine  under  examination.  Carefully  wash  out 
tube  A  with  water  and  introduce  into  it  sodium 
hypobromite  solution,^  being  careful  to  fill  the 
bulb  sufficiently  full  to  prevent  the  entrance 
of  air  into  the  graduated  portion.  Now  allow 
I  c.c.  of  urine-  to  flow  from  tube  B  into  tube  A, 
and  after  the  evolution  of  gas  bubbles  has  ceased 
(10-20  minutes)  take  the  reading  of  the  gradu- 
ated scale  on  tube  A. 

Calculation. — Observe  the  reading  on  the 
graduated  scale  of  tube  A.  This  tube  is  so 
graduated  as  to  represent  the  weight  of  urea,  in 
grams,  per  cubic  centimeter  of  urine.  If  w-e 
wish  to  compute  the  percentage  of  urea  present 
this  may  be  done  very  readily  by  simply  moving 
the  decimal  point  two  places  to  the  right;  e.g.,  if 
the  reading  is  0.02  gram  the  urine  contains  2 
per  cent  of  urea. 

Interpretation. — See  page  493. 

{b)  Hypobromite  Method  (using  Marshall's  Urea  Apparatus). — Place  the  thumb 
over  the  side  opening  of  the  bulbed-tube  of  the  apparatus  (Fig.  162)  and  carefully 
fill  the  tube  with  sodium  hypobromite  solution.'  Close  the  opening  in  the  end  of 
the  tube  with  a  rubber  stopper,  incline  the  tube  to  allow  air-bubbles  to  escape,  and 

*  Robinson  and  Muller:  Jonr.  Am.  Med.  Ass'n,  62,  514,  1914. 

^  If  the  content  of  urea  in  the  urine  under  examination  is  large,  tlie  urine  may  be  diluted 
with  water  before  determining  the  urea.  If  this  is  done  it  must  of  course  be  taken  into  con- 
sideration in  computing  the  content  of  urea. 

'  The  ingredients  of  the  sodium  hypobromite  solution  should  be  prepared  in  the  form  of 
two  separate  solutions.  When  needed  for  use  mi.\  one  volume  of  solution  a,  one  volume  of 
solution  b,  and  3  volumes  of  water. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams  of  bromine  and  make 
the  total  volume  of  the  solution  i  liter. 

(b)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of  i  .250.  This  is  approxi- 
mately a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles. 

32 


Fig 


I  60. DORF-MlS-HlNDS 

Ureometer. 


498 


PHYSIOLOGICAL   CHEMISTRY 


finally  invert  the  tube  and  fix  the  stoppered  end  in  the  saucer-shaped  vessel.  By 
means  of  the  graduated  pipette  rapidly  introduce  i  c.c.  of  urine^  into  the  hypo- 
bromite  solution  through  the  side  opening  of  the  bulbed-tube.  Withdraw  the 
pipette  immediately  after  the  urine  has  been  introduced.  When  the  decomposition 
of  the  urea  is  completed  (10-20  minutes),  gently  tap  the  bulbed-tube  with  the  finger 
in  order  to  dislodge  any  gas  bubbles  which  may  have  collected  on  the  inner  surface 
of  the  glass.  The  atmospheric  pressure  should 
now  be  equalized  by  attaching  the  funnel-tube 
to  the  bulbed-tube  at  the  side  opening  and 
introducing  hypobromite  solution  into  it  un- 
til the  columns  of  liquid  in  the  two  tubes  are 
uniform  in  height.  The  graduated  scale  of 
the  bulbed-tube  should  now  be  read  in  order 
to  determine  the  number  of  cubic  centimeters 
of  nitrogen  gas  evolved.  By  means  of  the  ap- 
pended formula  the  weight  of  the  urea  present 
in  the  urine  under  examination  may  be  com- 
puted. 


jTTTTTTnTIIIHIIIIIIIHIMI  i|i'|ni|||lllli;|i|li|l.'ihJilnl!-^^ 

Fro.  161. — Marshall  Urea  Apparatus. 
(Tyson.) 
a,  Bulbed  measuring  tube;   b,  saucer- 
shaped    vessel;    c,  graduated  pipette;   d, 
funnel-tube. 


Fig.  162. — HiJFNER  Urea  Apparatus. 


Calculation.^ — By  properly  substituting  in  the  following  formula,  the  weight  of 
urea,  in  grams,  contained  in  the  volume  of  urine  decomposed  (i  c.c.  or  more)  may 
readily  be  determined: 

1  Ordinarily  i  c.c.  of  urine  is  sufficient;  more  may  be  used,  however,  if  its  content  of  urea 
is  very  low. 

2  0.00366s  =  coefficient  of  expansion  of  gases  for  i°C.  354.5  =  number  of  c.c.  of  nitrogen 
gas  evolved  from  i  gram  of  urea. 


URINE 


499 


vjp  -  T) 

354.5X760(1+0.0036650 

IV  =  weight  of  urea,  in  grams. 

V  =  observed  volume  of  nitrogen  expressed  in  cubic  centimeters. 
p  =  barometric  pressure  expressed  in  millimeters  of  mercury. 

T  =  tension  of  aqueous  vapor^  for  temperature  /. 
t    =  temperature  (Centigrade). 

If  we  wish  to  calculate  the  percentage  of  urea  we  may  do  so  by  means  of  the 
following  proportion  in  which  y  represents  the  volume  of  urine  used  and  w  denotes 
the  weight  of  the  urea  contained  in  the  volume  y: 

y  :w  ::  X  :  {percentage  of  urea). 

Sodium  hypobromite  solution  may  also  be  employed  for  the  determination  of 
urea  in  the  apparatus  devised  by  Hiifner,  which  is  pictured  in  Fig.  162. 
Interpretation. — See  page  493. 

Ammonia 

I.  Folin's  Method. — Principle. — The  ammonia  of  the  urine  is  set 
free  bv  the  addition  of  an  alkali  and  this  ammonia  is  then  carried  over 


Fig.  163. — FoLiN  Ammonia  App.-\ratus. 

by  an  air  current  into  a  flask  containing  a  measured  amount  of  standard 
acid.  The  excess  acid  is  then  titrated.  The  necessity  for  distillation 
is  avoided. 

^The  values  of  T  for  the  temperatures  ordinarily  met  with  are  given  in  the  following 
table: 


Temp.  Tension  in  mm. 

IS°C 12.677 

i6°C 13519 

i7°C 14.009 

i8°C 15.351 

19  (^' 16-345 

20°C 17-396 


Temp.  Tension  in  mm. 

2i°C 18.505 

22°C 19-675 

23°C 20.909 

24°C 22.311 

25'C 23.582 


500 


PHYSIOLOGICAL   CHEMISTRY 


Procedure. — Place  25  c.c.  of  urine  in  an  aerometer  cylinder,  30-40  cm.  in 
height  (Fig.  163,  p.  499),  add  about  i  gram  of  dry  sodium  carbonate  and  introduce 
some  crude  petroleum  to  prevent  foaming.  Insert  into  the  neck  of  the  cylinder  a 
rubber  stopper  provided  with  two  perforations,  into  each  of  which  passes  a  glass 
tube,  one  of  which  reaches  below  the  surface  of  the  Uquid.  The  shorter  tube 
(10  cm.  in  length)  is  connected  with  a  calcium  chloride  tube  filled  with  cotton,  and 
this  tube  is  in  turn  joined  to  a  glass  tube  extending  to  the  bottom  of  a  500  c.c. 
wide-mouthed  flask  which  is  intended  to  absorb  the  ammonia  and  for  this  pur- 
pose should  contain  20  c.c.  of  N/io  sulphuric  acid,  200  c.c.  of  ammonia-free 
distilled  water  and  a  few  drops  of  an  indicator  (alizarin  red 
or  Congo  red).  To  insure  the  complete  absorption  of  the 
ammonia  the  absorption  flask  is  provided  with  a  Folin  im- 
proved absorption  tube  (Fig.  164),  which  is  very  effec- 
tive in  causing  the  air  passing  from  the  cylinder  to  come 
into  intimate  contact  with  the  acid  in  the  absorption  flask. 
In  order  to  exclude  any  error  due  to  the  presence  of  am- 
monia in  the  air  a  similar  absorption  apparatus  to  the  one 
just  described  is  attached  to  the  other  side  of  the  aerom- 
eter cylinder,  thus  insuring  the  passage  of  ammonia-free 
air  into  the  cylinder.  With  an  ordinary  filter  pump  and 
good  water  pressure  the  last  trace  of  ammonia  should  be 
removed  from  the  cylinder  in  about  one  and  one-half  hours. ^ 
The  number  of  cubic  centimeters  of  the  N/io  sulphuric  acid 
neutraUzed  by  the  ammonia  of  the  urine  may  be  determined 
by  direct  titration  with  N/io  sodium  hydroxide. 

Steele^  has  suggested  a  modification  for  use  on 
urines  containing  triple  phosphate  sediments.  In 
this  modification  0.5-1.0  gram  of  NaOH  and 
about  15  grams  of  NaCl  are  substituted  for  the 
Na2C03  of  the  Folin  method.  The  use  of  sodium 
hydroxide  and  chloride  instead  of  carbonate  has  also  been  recom- 
mended by  other  workers^  as  a  general  procedure,  inasmuch  as  triple 
phosphate  crystals  are  almost  always  formed  on  adding  sodium  car- 
bonate and  these  are  decomposed  with  some  difficulty  by  sodium 
carbonate  but  readily  by  the  hydroxide.  It  has  not  been  shown 
that  the  use  of  sodium  hydroxide  in  this  manner  brings  about  the 
decomposition  of  any  other  urinary  nitrogen  compounds. 


Fig.  164. — Folin 
Improved  Absorp- 
tion Tube. 


Calculation. — Subtract  the  number  of  cubic  centimeters  of  N/io  sodium 
hydroxide  used  in  the  titration  from  the  number  of  cubic  centimeters  of  N/io  sul- 
phuric acid  taken.  The  remainder  is  the  number  of  cubic  centimeters  of  N/io 
sulphuric  acid  neutralized  by  the  NH3  of  the  urine.     One  c.c.  of  N/io  sulphuric 

^  With  any  given  filter  pump  a  "  check  "  test  should  be  made  with  urine  or,  better,  with  a 
solution  of  an  ammonium  salt  of  known  strength  to  determine  how  long  the  air  current  must 
be  maintained  to  remove  all  the  ammonia  from  25  c.c.  of  the  solution. 

^Steele:  Jour.  Biol.  Chetn.,  8,  365,  1910. 

^Benedict  and  Ostcrberg:  Biochem.  Bull.,  3,  41,  1913. 
Shulansky  and  Gies:  Biochem.  Bull.,  3,  45,  1913. 


URINE  501 

acid  is  equivalent  to  0.0017  gram  of  NH3.  Therefore  if  y  represents  the  volume 
of  urine  used  in  the  determination  and  y'  the  number  of  cubic  centimeters  of 
N/io  sulphuric  acid  neutralized  by  the  NH3  of  the  urine,  we  have  the  following 
proportion : 

y'  :  ioo::y'Xo.ooi7:x  (percentage  of  NH3  in  the  urine  examined). 

Calculate  the  quantity  of  NH3  in  the  24-hour  urine  specimen. 

Interpretation. — The  average  daily  output  of  ammonia  in  the  urine 
is  about  0.7  gram,  amounting  to  2.5-4.5  per  cent  of  the  total  nitrogen 
excretion.  It  is  increased  by  the  ingestion  of  acids  or  acid-forming 
foods  and  decreased  by  the  ingestion  of  alkalis  or  base-forming  foods. 
In  acidosis  it  may  be  very  greatly  increased,  being  excreted  in  com- 
bination with  hydro-oxybutyric  and  other  acids.  Values  of  5  grams 
have  been  noted.  It  is  at  the  same  time  increased  relative  to  total 
nitrogen  and  urea.  In  pronounced  liver  disorders  the  same  thing  is 
noted,  as  ammonia  is  not  so  completely  transformed  into  urea  before 
excretion. 

2.  Micro  -chemical  Method  of  Folin  and  MacCallum.  ^ — Principle. — 
This  method  is  a  combination  of  the  aeration  procedure  for  ammonia 
with  its  colorimetric  determination  by  means  of  Nessler-Winkler  solu- 
tion. It  gives  satisfactory  results,  but  is  probably  not  as  accurate  as 
the  regular  Fohn  procedure  where  the  amount  of  substance  for  analysis 
is  not  limited. 

Procedure. — ^By  means  of  Ostwald  pipettes  introduce  1-5  c.c.  of  urine- 
into  a  Jena  test-tube  (20-25  mm.  by  200  mm.)  and  add  to  the  urine  a  few  drops 
of  a  solution  containing  10  per  cent  of  potassium  carbonate  and  15  per  cent  of 
potassixnn  oxalate.  To  prevent  foaming  add  a  few  drops  of  kerosene  or  heavy, 
crude  machine  oil.  Pass  a  strong  air  current  (see  page  487)  through  the  mixture 
imtil  the  ammonia  has  been  entirely  removed.'  Collect  the  ammonia  in  a  100 
c.c.  volumetric  flask  containing  about  20  c.c.  of  ammonia-free  water  and  2  c.c. 
of  N/io  acid. 

Nesslerize  as  described  in  the  method  for  total  nitrogen,  page  488,  and  com- 
pare with  I  mg.  of  nitrogen  obtained  from  a  standard  ammoniimi  sulphate  solu- 
tion and  similarly  Nesslerized. 

It  has  been  noted  that  a  trace  of  something  capable  of  giving  a  color  with 
the  Nessler-Winkler  solution  continues  to  come  long  after  all  the  ammonia 
has  been  removed  from  the  urine.  The  nature  of  this  substance  has  not  yet 
been  determined.  In  actual  determinations  by  this  method,  the  influence  of 
this  unknown  substance,  because  of  the  small  volume  of  urine  used,  is  entirely 
negUgible. 

1  Folin  and  MacCallum:  Jour.  Biol.  Chcin.,  ii,  523,  1912. 

^The  volume  of  urine  taken  should  contain  0.75-1.5  mg.  of  ammonia  nitrogen.  With 
normal  urines  2  c.c.  will  generally  yield  the  desired  amount.  With  very  dilute  urines  5  c.c. 
may  be  required,  while  with  diabetic  urines  rich  in  ammonium  salts  i  c.c.  may  be  excessive^ 
thus  requiring  dilution. 

^  Ordinarily  a  period  of  ten  minutes  is  suflSciently  long. 


502  PHYSIOLOGICAL   CHEMISTRY 

3.  Formol  Titration  Method  (Malfatti).^ — Principle. — This  method 

is  based  on  the  reaction  taking  place  when  formalin  solution  is 
added  to  a  solution  containing  ammonium  salts  (see  Amino-acid 
Nitrogen,  below).  An  acid  reaction  is  produced  in  the  mixture, 
which  is  then  titrated  with  standard  alkaH  using  phenolphthalein  as 
an  indicator.  Amino-acids  give  the  same  reaction  so  that  the  result 
of  the  titration  represents  ammonia  +  amino-acid  nitrogen.  This 
method  may  be  used  for  the  rapid  cHnical  estimation  of  these  forms  of 
nitrogen  as  a  substitute  for  an  ammonia  determination,  but  the  results 
do  not  represent  ammonia  as  is  sometimes  stated. 

Procedure. — To  25  c.c.  of  urine  in  a  200  c.c.  Erlenmeyer  flask  add  15-20 
grams  of  finely  pulverized  potassiiim  oxalate,  a  few  drops  of  phenolphthalein, 
and  titrate  to  a  faint  but  permanent  pink  color  with  N/'io  NaOH.  (The  urine 
mixture  just  after  neutralization  in  the  urinary  acidity  determination  (see  page 
479)  may  be  used.)  Then  add  10  c.c.  of  neutral  formalin  solution  (see  amino- 
acid  nitrogen),  mix  well  and  titrate  with  N/io  sodium  hydroxide  to  a  permanent 
pink  color. 

Calculation. — One  c.c.  of  N/'io  sodivmi  hydroxide  is  equivalent  to  1.7 
mg.  of  ammonia.  Multiply  the  nvunber  of  cubic  centimeters  of  N/io  alkali 
used  by  1.7  and  by  4  to  get  the  number  of  milligrams  of  ammonia  +  amino- 
acid  nitrogen  (expressed  as  ammonia)  in  100  c.c.  of  the  urine  examined. 

Amino-Acid  Nitrogen 

I.  Henriques-Sorensen  Formol  Titration  Method.- — Principle. — 
A  solution  containing  amino-acids  is  nearly  neutral  in  reaction.  If 
formaldehyde  be  added,  however,  the  following  reaction  takes  place 
with  the  formation  of  methylene  derivatives  which  an  more  strongly  acid 
in  reaction  due  to  the  destruction  of  the  basic  properties  of  the  amino 
groups.  The  carboxyl  groups  may  then  be  titrated  using  phen- 
olphthalein as  an  indicator. 

R.CH.NH2 

I  +  CH2O  =  R— CH— N:  CH2  +  H2O. 

COOH  I 

COOH 

The  acidity  as  shown  by  the  titration  is  a  measure  of  the  amount  of 
amino-acid  nitrogen  present.  Ammonia  likewise  reacts  with  formalde- 
hyde in  a  siinilar  manner  as  is  shown  in  the  following  equation: 

4NH4Cl-h6CH20  =  N4(CH2)6+6H20-f4HCl. 

Hence  the  formol  titration  in  the  presence  of  ammonia  gives  results 
which  include  both  amino-acid  and  ammonia  nitrogen.     Ammonia 

'  Malfatti:  Z.  anal.  Chem.,  47,  273,  1908. 

^Henriques  and  Sorensen:  Zeit.  physiol.  chem.,  64,  120,  igog. 


URIXE  503 

may  be  determined  and  a  correction  applied,  or  the  ammonia  may  be 
removed  by  means  of  phosphotungstic  acid.  Phosphates  also  inter- 
fere by  obscuring  the  end-point  and  are  removed  by  the  addition  of 
barium  salts. 

It  must  be  borne  in  mind  that  polypeptides  and  still  more  complex 
protein  derivatives  likewise  react  with  formol  to  a  certain  degree  so 
that  the  results  do  not  strictly  represent  "amino-acid  nitrogen." 

The  method  is,  with  some  modifications  involving  the  preparation 
of  the  solution  to  be  titrated,  applicable  in  the  determination  of  amino- 
acids  in  any  medium,  e.g.,  urine,  protein  digests,  etc.  When  poorly 
dissociated  acids,  e.g.  some  fatty  acids,  are  present,  these  will  in  part 
be  included  in  the  result  and  lead  to  values  which  are  too  high.  Certain 
of  the  amino-acids  when  present  in  large  amounts  will  give  erroneous 
results,  but  in  the  ordinary  urine  or  digest  these  errors  are  either 
negligible  or  compensate  each  other.  In  the  titration  of  colored  solu- 
tions the  control  solution  which  is  necessary  in  this  method  must  be 
colored  to  correspond  with  the  color  of  the  unknown  solution. 

Procedure. — The  determination  of  the  amino-acids  is  carried  out  as  follows : 
The  solution  to  be  analyzed,  if  carbonates,  phosphates  and  ammonia  are  absent, 
is  made  neutral  to  litmus  (paper)  and  the  solution  titrated  with  formalde- 
hyde as  below.  1  In  case  carbonates,  phosphates  or  ammonia  are  present  a 
preliminary  treatment  is  necessary  which  will  vary  according  to  the  quantity 
of  ammonia  present. 

(a)  For  Small  Amounts  of  Ammonia. — Applicable  to  most  urines.  Fifty 
c.c.  of  the  material  under  examination  is  pipetted  into  a  100  c.c.  measuring 
flask  and  i  c.c.  phenolphthalein  solution-  and  2  grams  of  solid  barium  chloride 
are  added;  the  whole  is  shaken,  to  saturate  the  solution  with  barium  chloride; 
saturated  barium  hydroxide  solution  is  added  until  the  red  color  of  the  phenol- 
phthalein develops  and  then  an  excess  of  5  c.c.  is  added.  The  flask  is  filled 
to  the  graduation  mark  with  water,  shaken  and  permitted  to  stand  for  15  minutes, 
after  which  it  is  filtered  through  a  dry  filter.  Eighty  c.c.  of  the  clear  red  filtrate 
iwhich  corresponds  to  40  c.c.  of  the  liquid  under  examination!  are  placed  in 
a  100  c.c.  measuring  flask,  neutraUzed  to  Utmus  and  diluted  to  100  c.c.  with 
freshly  boiled  water.  Equal  portions  of  this  solution,  40  c.c.  (equivalent  to 
16  c.c.  of  the  original  solution),  may  be  taken  for  analysis,  one  for  the  formol 
titration  and  the  other  for  the  determination  of  ammonia  nitrogen.'' 

(b)  For  Large  Amounts  of  Ammonia. — After  the  treatment  with  phenol- 
phthalein, bariimi  chloride,  and  barium  hydroxide,  and  the  solution  has  been 
diluted  to  100  c.c.  as  in  (a)  above,  the  ammonia  is  distilled  off,  in  vacuo.* 

'  As  a  standard  of  comparison  the  litmus  paper  used  for  neutralization  ii  contrasted  with 
a  similar  piece  dipped  in  a  phosphate  solution  having  a  neutral  reaction  (M,  15  KHjPO^and 
M/is  NajHPO^). 

^  A  solution  of  0.5  gram  of  phenolphthalein  in  50  c.c.  of  alcohol  and  50  c.c.  of  water. 

'The  determination  of  ammonia  may  be  dispensed  with  in  case  a  separate  determina- 
tion is  made. 

*  For  particulars  with  regard  to  the  distillation,  etc.,  see  Henriques  and  Sorensen:  Zeit. 
physiol.  Client.,  64,  137,  igog. 


504  PHYSIOLOGICAL    CHEMISTRY 

In  case  the  solution  is  deeply  colored,  as  in  protein  digests,  it  may  be  neces- 
sary to  decolorize^  before  the  titration  is  attempted. 

Final  Titration. — For  the  final  titration  a  volume  of  from  20-40  c.c.  which  con- 
tains approximately  0.025  gram  of  nitrogen  is  the  most  desirable.  A  control 
solution  is  run  composed  of  an  equal  volume  of  boiled  distilled  water  and  20  c.c. 
of  the  formaldehyde  mixture.  ^  This  control  solution  is  colored^  so  that  its 
tint  matches  that  of  the  solution  to  be  titrated. 

To  this  control  is  added  about  half  the  volume  of  N  '5  alkali  which  will  be 
used  in  the  titration  of  the  solution  under  investigation  and  it  is  then  titrated 
with  N/5  acid  to  a  faint  red  (first  stage). ^ 

An  additional  drop  of  N/5  alkali  is  added,  which  imparts  a  distinct  red  to 
the  solution  (second  stage). 

The  solution  to  be  analyzed  is  now  titrated  to  the  color  produced  in  the 
second  stage  of  the  control.  The  formaldehyde  mixture  is  now  added;  10  c.c. 
for  each  20  c.c.  of  the  solution,  and  the  mixture  again  titrated  to  the  second 
stage  with  N/5  alkaU.^ 

Two  drops  of  the  N/5  alkali  are  now  added  to  the  control  solution  which 
assumes  a  deep  red  color  (third  stage).  Fifth  normal  alkali  is  now  added  to 
the  solution  under  examination  until  it  assumes  a  color  corresponding  to  the 
third  stage  of  the  control.     This  completes  the  titration. 

Calculation. — The  calculations  are  similar  to  those  which  pertain  to  any 
acidimetry  procedure.  Each  cubic  centimeter  of  an  N/5  alkali  or  acid  solution 
is  equivalent  to  0.0028  gram  of  nitrogen.  An  example  will  illustrate  the  pro- 
cedure: 40  c.c.  of  solution  (16  c.c.  of  urine)  required  5.10  c.c.  N/5  NaOH; 
control,  o.io  c.c.  N/5  NaOH;  total  required  for  amino-acids  5.00  c.c.  equivalent 
to  0.014  gram  of  nitrogen.  Ammonia  nitrogen  in  i6  c.c.  of  urine  0.007  gram  N. 
Then  0.014  —  0.007  =  0.007  gram  amino-acid  nitrogen  in  i6  c.c.  of  urine. 

Interpretation. — The  excretion  of  total  amino-acid  nitrogen  by  a 
normal  adult  averages  between  0.4  to  i.o  gram  per  day  or  from  2  to  6 
per  cent  of  the  total  nitrogen.  Free  amino-acid  nitrogen  (see  Van 
Slyke  procedure)  is  considerably  less  than  this,  ordinarily  0.5  to  i.o 
per  cent  of  the  total  nitrogen.  The  amount  may  be  largely  increased 
in  disorders  associated  with  tissue  waste  as  typhoid,  in  pronounced 
atrophy  of  the  liver,  acidosis,  etc. 

2.  Benedict-Murlin  Modification.^ — Principle. — In  this  method  the  ammonia 
is  removed  by  means  of  phosphotungstic  acid,  and  excess  acid  as  well  as  carbonates 
and  phosphates  carried  down  with  barium. 

^  For  methods  see  Jessen-Hansen,  Abderhalden's  Arbeits  Methoden,  vol.  6,  p.  202, 1912. 

2  The  formaldehyde  solution  is  freshly  prepared  for  each  set  of  determinations  as  follows: 
to  50  c.c.  of  commercial  formaldehyde  (formol)  (30-40  per  cent)  add  i  c.c.  of  the  phenol- 
phthalein  solution.  N/5  alkali  is  then  added  until  the  mixture  acquires  a  faint  red  color. 
The  volume  of  the  formaldehyde  used  will  vary  with  the  volume  of  the  solution  to  be  ana- 
lyzed; approximately  10  c.c.  of  the  formalin  solution  are  added  for  each  20  c.c.  of  the  un- 
known solution. 

^  Solution  of  Bismark  brown  is  very  satisfactory  for  urines.  Tropaeolin  O,  Tropaeolin 
00,  />-nitro-phenol,  methyl  orange  or  alizarin  sulphonate,  may  be  used. 

*  This  procedure  is  recommended  in  order  that  the  final  volume  of  the  control  and  the 
unknown  solutions  shall  be  approximately  the  same  when  the  process  is  complete. 

'  This  is  best  accomplished  by  adding  alkali  until  the  color  is  deeper  than  that  of  the 
control,  then  acid  again  until  lighter  and  finally  alkali  to  the  desired  color. 

*  Benedict  and  Murlin:  Jour.  Biol.  Cheni.,  16,  385,  1913. 


URINE  505 

Procedure. — Measure  into  a  500  c.c.  Erlenmeyer  flask  200  c.c.  of  a  24-hour 
urine  which  has  been  diluted  to  2000  c.c.  (or  its  equivalent).  Add  an  equal  volume 
of  10  per  cent  phosphotungstic  acid  (Merck) ^  in  2  per  cent  HCl.  Let  stand  at  least 
three  hours,  better  over  night.  Pour  off  250  c.c.  of  the  clear  fluid,  add  i  c.c.  of  a 
0.5  per  cent  solution  of  phenolphthalein  and  then  barium  hydroxide  in  substance 
until  the  whole  fluid  turns  decidedly  pink.  The  barium  hydroxide  should  be  added 
a  very  little  at  a  time.  Let  stand  one  hour.  Filter  off  two  100  c.c.  samples  (=50 
c.c.  urine).  Neutralize  these  samples  to  litmus  (using  good  quality  litmus  paper) 
with  N/5  HCl.  Add  at  once  10-20  c.c.  of  neutral  formalin-  and  titrate  cautiously 
to  a  deep  red  color,  i.e.,  until  the  drop  produces  no  additional  color  with  N/io 
NaOH.  Deduct  from  the  result  thus  obtained  the  amount  of  N/io  NaOH  neces- 
sary to  produce  the  same  depth  of  color  in  an  equal  quantity  of  water,  freed  from 
carbon  dioxide  by  boiling  and  cooling,  and  to  which  an  equal  volume  of  neutral 
formalin  has  been  added. 

Calculation. — One  c.c.  of  N/io  NaOH  is  equivalent  to  1.4  mg.  of  amino-acid 
nitrogen.  Multiply  the  number  of  cubic  centimeters  of  N/io  NaOH  used  (after 
deducting  for  control  as  indicated  above)  by  1.4  and  by  2  (as  the  equivalent  of  50 
c.c.  of  urine  was  used)  to  obtain  the  number  of  milligrams  of  amino-acid  nitrogen 
in  100  c.c.  of  the  urine. 

Interpretation. — See  page  504. 

3.  Method  of  Frey-Gigon,^ — Principle. — The  ammonia  is  removed  from  the 
urine  by  aspiration  after  treatment  with  barium  hydroxide  and  the  formol  titration 
performed  in  the  usual  manner. 

Procedure. — Treat  50  c.c.  of  urine  in  an  aerometer  cylinder  such  as  used  in  the 
Folin  ammonia  method,  with  20  c.c.  of  saturated  barium  hydroxide  solution  and 
15  c.c.  of  alcohol.  Aspirate  for  two  or  three  hours,  using  a  slow  current  of  air. 
The  ammonia  is  carried  off.  (It  may  be  collected  in  standard  acid  solution  and 
determined  as  in  the  Folin  method  (see  page  499)  if  desired.)  Transfer  the  urine 
mixture  quantitatively  to  a  250  c.c.  flask  and  make  to  mark  with  distilled  water. 
Shake  well,  allow  to  settle,  filter.  Take  100  c.c.  of  the  filtrate  and  just  neutralize 
with  N/5  hydrochloric  acid,  using  rosolic  acid  as  an  indicator.  Then  to  another 
100  c.c.  portion  of  the  filtrate  add  an  amount  of  the  standard  acid  equal  to  that 
added  to  the  first  portion.  Next  add  10  c.c.  of  neutral  formalin  solution,  a  few 
drops  of  phenolphthalein  and  titrate  in  the  usual  manner  to  a  red-violet  color. 
Calculate  as  in  the  preceding  method. 

The  amino-acid  nitrogen  may  also  be  appro.ximately  determined  by  carr>'ing 
out  the  titration  for  ammonia  +  and  amino-acid  nitrogen  as  given  under  Ammonia, 
page  502,  making  a  separate  determination  of  ammonia,  and  subtracting  the  latter 
result  from  the  former. 

4.  Van  Slyke's  Method  for  Total  Amino-Acid  Nitrogen.*— Take  25  c.c.  of 
urine^  and  mix  with  i  c.c.  of  concentrated  sulphuric  acid  and  heat  in  an  auto- 
clave at  180°  (oil  bath  temperature)  for  one  and  one-half  hours.  Transfer  to  a 
50  c.c,  flask  and  add  2  grams  powdered  calcium  hydroxide.  Shake  thoroughly, 
make  up  to  50  c.c.  and  filter  through  a  dry  folded  filter.     Transfer  20  c.c.  of  the 

'  Kahlbaum's  preparation  is  a  very  different  substance. 

-  To  50  c.c.  commercial  formalin  solution  (30-40  per  cent)  add  i  c.c.  of  phenolphthalein 
solution  and  then  N/s  NaOH  to  a  very  faint  pink  color.  The  solution  should  be  freshly 
prepared. 

•'  Prey  and  Gigon:  Biochem.  Zeil.,  22,  309,  1909. 

*  Van  Slyke:  Jour.  Biol.  Chcm.,  16,  125,  1913. 

'See  (Van  Slyke:  Proc.  Soc.  Exp.  Biol,  and  Med.,  13,  63,  1915)  for  treatment  of  urines 
containing  glucose  or  albumin. 


5o6  PHYSIOLOGICAL   CHEMISTRY 

filtrate  to  a  Jena  glass  evaporating  dish  and  concentrate  to  dryness  on  the  water- 
bath.  This  requires  about  half  an  hour.  The  residue  is  moistened  with  i  c.c. 
of  50  per  cent  acetic  acid  to  bring  the  calcium  hydroxide  and  carbonate  into 
solution,  and  is  then  washed  into  a  10  c.c.  flask  and  filled  up  to  the  mark.  One 
can  use  the  entire  solution  for  determination  of  the  amino -nitrogen  in  the  large 
amino -apparatus,  or  use  2  c.c.  portions  for  the  micro -apparatus.  (See  Van 
Slyke  Apparatus,  Figs.  34  and  35,  p.  88  in  Chapter  IV  on  Proteins.) 

The  length  of  time  which  the  nitrous  acid  solution  should  be  shaken  in  order 
to  drive  off  all  the  amino-nitrogen  depends  somewhat  on  the  temperature. 
When  the  latter  is  15-20°  the  time  should  be  five  to  four  minutes;  for  §0-25°  it 
is  three  minutes,  for  25-30°,  two  and  a  half  to  two  minutes.  It  is  preferable 
that  the  solution  should  be  shaken  vigorously  with  a  motor  and  the  time  kept 
down  to  these  limits,  for  the  sake  not  only  of  rapidity  but  of  accuracy. 

Van  Slyke's  Method  for  Free  Amino-Acid  Nitrogen. — To  25  c.c.  of  urine^ 
in  a  50  c.c.  flask  add  urease  solution  and  allow  to  stand  for  one  and  one-half 
times  the  interval  which  has  been  found  necessary  to  effect  the  maximimi  de- 
composition of  urea,  as  observed  by  titration  of  the  ammonia.  The  last  traces 
of  urea  are  decomposed.  At  the  end  of  the  digestion  period  10  c.c.  of  a  10  per 
cent  suspension  of  calcium  hydroxide  are  added,  the  mixture  shaken  and  made 
up  to  50  c.c.  Then  filter,  evaporate,  and  complete  the  determination  according 
to  the  method  outhned  under  total  amino-acid  nitrogen,  above. 

Creatinine 

Folin's  Colorimetric  Method. — Principle.- — This  method  is  based 
upon  the  characteristic  property  possessed  by  creatinine,  of  yielding  a 
certain  definite  color-reaction  in  the  presence  of  picric  acid  in  alkaline 
solution. 

Procedure. — Place  10  c.c  of  urine  in  a  500  c.c.  volxometric  flask,  add  15  cc.  of  a 
saturated  solution  of  picric  acid  and  5  c.c.  of  a  10  per  cent  solution  of  sodium  hy- 
droxide, shake  thoroughly  and  allow  the  mixture  to  stand  for  five  minutes.  During 
this  interval  pour  a  little  N/2  potassiimi  bichromate  solution^  into  each  of  the  two 
cylinders  of  the  colorimeter  (Duboscq's,  see  Fig.  153,  p.  486)  and  carefully  adjust 
the  depth  of  the  solution  in  one  of  the  cylinders  to  the  8  mm.  mark.  A  few  prelimi- 
nary colorimetric  readings  may  now  be  made  with  the  solution  in  the  other  cylinder, 
in  order  to  insure  greater  accuracy  in  the  subsequent  examination  of  the  solution  of 
unknown  strength.  Obviously  the  two  solutions  of  potassium  bichromate  are 
identical  in  color  and  in  their  examination  no  two  readings  should  differ  more  than 
0.I-0.2  mm.  from  the  true  value  (8  mm.).  Four  or  more  readings  should  be  made 
in  each  case  and  an  average  taken  of  all  of  them  exclusive  of  the  first  reading,  which 
is  apt  to  be  less  accurate  than  the  succeeding  readings.  In  time  as  one  becomes 
proficient  in  the  technic  it  is  perfectly  safe  to  take  the  average  of  the  first  two 
readings. 

At  the  end  of  the  five-minute  interval  already  mentioned,  the  contents  of  the 
500  c.c.  flask  are  diluted  to  the  500  c.c.  mark,  the  bichromate  solution  is  thoroughly 
rinsed  out  of  one  of  the  cyclinders  and  replaced  with  the  solution  thus  prepared  and 
a  number  of  colorimetric  readings  are  immediately  made. 

1  See  note  5,  page  505. 

''This  solution  contains  24.55  grams  of  potassium  bichromate  to  the  liter  A  pure 
creatinine  standard  is  to  be  preferred,  see  p.  507. 


URINE  507 

Ordinarily  10  c.c.  of  urine  is  used  in  the  determination  by  this  method,  but  if 
the  content  of  creatinine  is  above  15  mg.  or  below  5  mg.  the  determination  should 
be  repeated  with  a  volume  of  urine  selected  according  to  the  content  of  creatinine. 
This  variation  in  the  volume  of  urine  according  to  the  content  of  creatinine  is  quite 
essential,  since  the  method  loses  in  accuracy  when  more  than  15  mg.  or  less  than 
5  mg.  of  creatinine  is  present  in  the  solution  of  unknown  strength. 

Calculation.  By  experiment  it  has  been  determined  that  10  mg.  of  pure  crea- 
tinine, when  brought  into  solution  and  diluted  to  500  c.c.  as  explained  in  the  above 
method,  yields  a  mixture  8.1  mm.  of  which  possesses  the  same  colorimetric  value 
as  8  mm.  of  a  N/  2  solution  of  potassium  bichromate.  Bearing  this  in  mind  the 
computation  is  readily  made  by  means  of  the  following  proportion  in  which  y  repre- 
sents the  number  of  millimeters  of  the  solution  of  unknown  strength  equivalent  to 
the  8  mm.  of  the  potassiimi  bichromate  solution : 

y  :8.i  : :  10  :  X  (mg.  of  creatinine  in  the  quantity  of  urine  used). 

This  proportion  may  be  used  for  the  calcvdation  no  matter  what  volvune  of 
urine  (5,  10,  or  15  c.c.)  is  used  in  the  determination.  The  10  represents  10  mg.  of 
creatinine  which  gives  a  color  equal  to  8.1  mm.,  whether  dissolved  in  5, 10,  or  15  c.c. 
of  fluid. 

Calculate  the  quantity  of  creatinine  in  the  24-hour  urine  specimen. 

Interpretation. — The  daily  excretion  of  creatinine  by  an  adult  of 
medium  weight  averages  about  1^:4  grams.  The  value  is  nearly  con- 
stant from  day  to  day  for  a  given  individual  being  influenced  by  the  diet 
hardly  at  all  unless  this  contains  much  preformed  creatinine  (as  in  case 
of  a  heavy  meat  diet) .  The  excretion  of  creatinine  is  to  a  certain  extent 
a  measure  of  muscular  efficiency  and  of  the  amount  of  active  muscle 
tissue  in  the  body.  Relative  to  body  weight  less  creatinine  is  excreted 
by  obese  persons. 

Creatinine  excretion  is  decreased  in  disorders  associated  with  mus- 
cular atrophy  and  muscular  weakness.  It  increases  with  increased 
tissue  cetabolism  as  in  fever. 

By  the  "creatinine  coefl&cient"  is  meant  the  number  of  milligrams 
of  creatinine  — ////rogc«  excreted  daily  per  kilo  of  body  weight.  This 
varies  under  normal  conditions  from  7-1 1. 

Use  of  Pure  Creatinine  Standards. — Instead  of  using  as  a  standard  a  potassium 
dichromate  solution  as  above  indicated,  a  solution  of  pure  creatinine  is  to  be  recom- 
mended. By  using  this  certain  arbitrary  factors  are  eliminated  and  the  method 
becomes  of  more  general  applicability.  The  standard  need  not  be  set  at  a  definite 
mark  as  is  necessary  in  the  case  of  dichromate  and  temperature  and  time  have  less 
inlluence  on  the  accuracy  of  the  results.  A  stock  solution  of  pure  creatinine  (made 
according  to  Benedict's  directions;  see  Chapter  XXII  on  Physiological  Constituents 
of  Urine)  is  made  by  dissolving  i  gram  of  the  substance  in  sutVicient  N/ 10  HCl  to 
make  a  liter.  This  solution  contains  i  mg.  of  creatinine  per  cubic  centimeter.  In 
carrying  out  the  determination  treat  10  c.c.  of  the  stock  solution  in  the  same  \vay  and 
at  the  same  time  as  the  10  c.c.  sample  of  urine.  Compare  in  the  colorimeter.  The 
calculation  s  simple.     The  reading  of  the  standard  divided  by  the  reading  of  the 


5o8  PHYSIOLOGICAL   CHEMISTRY 

urine  gives  directly  the  number  of  milligrams  of  creatinine  per  cubic  centimeter  of 
urine. 

Folin's  Microchemical  Modification.' — Principle. — The  principle  is  the  same 
as  that  of  the  original  colorimetric  method  (see  page  506).  This  procedure  is  to  be 
recommended  particularly  where  only  small  amounts  of  material  are  available. 

Procedure. — One  c.c.  of  the  standard  creatinine  (see  above)  solution  (i  mg.  per 
c.c.)  is  measured  into  a  100  c.c.  volumetric  flask  and  i  c.c.  of  urine  into  another; 
20  c.c.  of  saturated  picric  acid  solution  (measured  with  a  cylinder)  are  added  to 
each  and  then  1.5  c.c.  of  a  10  per  cent  solution  of  sodium  hydroxide.  At  the  end 
of  ten  minutes  the  flasks  are  fiUed  up  to  the  mark  with  tap  water  and  the  color 
of  the  unknown  is  determined.  The  reading  of  the  standard  divided  by  the 
reading  of  the  unknown  gives  directly  the  number  of  milligrams  of  creatinine  in 
the  amount  of  urine  taken  for  analysis. 

3.  Shaffer's  Modification  for  the  Determination  of  Creatinine  in  Very  Dilute 
Solutions. 2 — The  regular  Folin  procedure  is  not  accurate  when  applied  to  urines 
containing  less  than  20  mg.  of  creatinine  per  100  c.c.  By  a  slight  modification  it 
becomes  applicable  to  creatinine  solutions  containing  as  little  as  i  mg.  or  less  per 
100  c.c. 

Procedure. — To  the  solution  under  examination  add  an  equal  volume  of  satu- 
rated picric  acid  solution  and  one-tenth  this  volume  of  10  per  cent  sodium  hydroxide 
solution.  After  standing  6-10  minutes  the  liquid  is  diluted  to  a  definite  volume 
depending  upon  the  intensity  of  the  color  developed.  With  very  dilute  solutions 
one  may  add  solid  picric  acid  equivalent  to  half  saturation  (0.6  per  cent)  and  when 
dissolved,  one-twentieth  the  volume  of  sodium  hydroxide.  Provided  the  creatinine 
solution  itself  has  not  sufficient  color  to  interfere,  the  results  by  this  method  appear 
to  be  as  accurate  as  the  original  procedure.  The  colorimetric  readings  and  calcu- 
lations are  made  in  the  same  way  as  in  the  preceding  methods. 

Creatine 

Folin-Benedict  Method.^ — Principle. — Creatinine  on  boiling  with 
acid  is  transformed  into  creatinine.  By  determining  the  content  of 
creatinine  before  and  after  the  acid  treatment  we  are  able  to  calculate 
the  amount  of  creatinine  originally  present  in  the  urine.  The  Folin 
colorimetric  method  (page  506)  is  used  for  determining  the  creatinine 
in  both  cases.     The  method  is  not  applicable  to  diabetic  urines. 

Procedure. — Introduce  into  a  small  flask  or  beaker  10  c.c.  of  the  urine  to 
be  examined.  (If  10  c.c.  contains  more  than  12  or  less  than  7  mg.  of  total 
creatinine  use  a  correspondingly  smaller  or  larger  volmne  of  urine.)  Add  from 
10-20  c.c.  of  normal  HCl,  and  a  pinch  or  two  of  powdered  or  granulated  lead. 
Boil  the  mixure  over  a  free  flame  as  slowly  or  as  rapidly  as  may  be  desired,  until 
very  nearly  down  to  dryness,  when  the  heating  should  be  continued  to  dryness 
either  on  the  water-bath  or  very  easily  by  simply  holding  the  vessel  in  the  hand 
and  heating  carefully  for  a  moment  or  two.  Let  the  residue  stand  on  the  water- 
bath  for  a  few  minutes  until  most  of  the  excess  of  hydrochloric  acid  gas  has  been 

'  Folin:  Jour.  Biol.  Client.,  17,  469,  1914. 
-Shaffer:  Jour.  Biol.  Chem.,  18,  525,  1914. 
^Benedict:  Jour.  Biol.  Chem.,  18,  191,  1914. 


URINE  509 

expelled,  after  which  dissolve  it  in  about  10  c.c.  of  hot  water  and  rinse  the  solu- 
tion quantitatively  through  a  plug  of  cotton  or  glass  wool  (to  remove  aU  metallic 
lead)  into  a  500  c.c.  volumetric  flask.  Add  20-25  c.c.  of  a  saturated  picric  acid 
solution  and  about  7-8  c.c.  of  a  10  per  cent  NaOH  solution,  which  contains  5 
per  cent  of  Rochelle  salt.'  At  the  end  of  five  minutes  fill  to  the  mark  with  water 
and  read  in  the  colorimeter  just  as  in  the  case  of  creatinine  (see  page  5061. 

Calculation. — Calculate  the  creatinine  content  of  the  solution  in  the  same 
manner  as  given  under  Creatinine  fpage  507 ) .  From  the  value  thus  obtained  sub- 
tract the  value  for  the  creatinine  content  of  the  urine  before  dehydration.  The 
difference  will  be  the  creatine  content  of  the  original  urine  in  terms  of  creatinine. 

Interpretation. — Creatine  occurs  only  in  very  small  amounts  in  the 
urine  of  normal  adults,  but  is  found  in  larger  amounts  in  that  of  children 
(10  to  56  mg.  per  day).  Creatine  ingestion  in  adults  has  little  effect 
on  the  urinary  excretion.  In  fasting,  the  amount  is  markedly  increased 
(it  may  amount  to  100  mg.  or  more  per  day).  Creatine  also  appears 
in  the  urine  after  high  water  ingestion.  It  is  found  in  many  pathological 
conditions  associated  with  malnutrition  and  disintegration  of  muscular 
tissue,  in  fever,  etc.  Very  large  amounts  have  been  found  in  cases  of 
carcinoma  of  the  liver. 

2.  Folin -Benedict  and  Myers  Method. ^ — To  20  c.c.  of  urine  in  a  50  c.c.  volu- 
metric flask,  add  20  c.c.  of  normal  hydrochloric  acid  and  place  the  flask  in  an  auto- 
clave at  a  temperature  of  117-120°  C.  for  one-half  hour.  Add  distilled  water  until 
the  volume  of  the  acid-urine  mixture  is  exactly  50  c.c,  close  the  flask  by  means  of 
a  stopper,  and  shake  it  thoroughly.  Approximately  neutralize  25  c.c.  of  this  mix- 
ture, introduce  it  into  a  500  c.c.  volumetric  flask  and  determine  its  creatinine  con- 
tent according  to  Folin's  Colorimetric  Method  (see  page  506). 

For  calculation  and  interpretation  see  the  foregoing  method. 

3.  Method  of  Folin. ^ — Water-hath  Procedure. — Heat  10  c.c.  of  urine  with  5  c.c. 
of  normal  hydrochloric  acid  on  the  boiling  water-bath  or  at  90°C.  for  three  hours. 
The  creatine  is  transformed  into  creatinine.  Some  darkening  takes  place  but  this 
does  not  interfere  because  of  the  subsequent  dilution.  The  mixture  is  made  up  to 
50  c.c,  25  c.c.  of  this  is  taken,  neutralized,  and  creatinine  plus  creatine  determined 
just  as  in  the  case  of  creatinine  alone.  The  creatine  is  obtained  by  difi^erence. 
This  procedure  may  be  used  for  diabetic  urines  which  is  not  the  case  with  the  auto- 
clave procedure  nor  with  the  Benedict  modification.  It  is  perhaps  not  quite  so 
accurate  as  the  autoclave  procedure. 

4.  Microchemical  Modification  of  Folin.* — By  greatly  diluting  the  urine  the 
time  required  for  the  conversion  of  creatine  to  creatinine  is  decreased,  and  picric 
acid  can  be  substituted  for  mineral  acid. 

Procedure. — Enough  urine  to  give  0.7-1.5  mg.  of  creatinine  is  measured  into 
a  weighed  Erlenmeyer  Jena  flask  (capacity  200  c.c);  20  c.c.  of  saturated  picric  acid 
solution,  about  130  c.c.  of  water,  and  a  few  very  small  pebbles  to  promote  even 
boiling  are  added  and  the  mixture  is  gently  boiled,  preferably  over  a  micro-burner 

'  The  Rochelle  salt  should  be  present  to  prevent  any  formation  of  turbidity,  which 
otherwise  may  occur,  due  to  the  presence  of  traces  of  dissolved  lead. 
-  Benedict  and  ^Iyers:  -Iw.  .T .  Phys.,  18,  397,  1907. 
^  Folin:  Zeilscttr,  f.  physiol.  Ckem.,  41,  222,  1904. 
*  Folin:  Jour.  Biol.  Clum.,  17,  469,  1914. 


lO  PHYSIOLOGICAL   CHEMISTRY 


for  about  one  hour.  At  the  end  of  this  time  the  heat  is  increased  and  the  solution 
is  boUed  down  to  rather  less  than  20  c.c.  The  flask  is  transferred  to  the  scales 
and  enough  water  is  added  to  make  the  total  solution  equal  to  20-25  grams.  The 
solution  is  cooled  in  running  water,  1.5  c.c.  of  10  per  cent  sodium  hydroxide  are 
added,  and  the  total  creatinine  is  determined  as  in  the  preformed  creatinine  deter- 
mination using  I  mg.  of  creatinine  as  a  standard.  The  method  has  been  found  to 
give  good  results  in  the  presence  of  glucose  and  other  sugars. 

Morris^  has  suggested  that  in  the  case  of  diabetic  urines  the  total  creatinine 
be  determined  after  precipitation  of  the  creatine  and  creatinine  with  picric  acid. 
The  method  is  not  recommended  as  a  regular  procedure. 

Uric  Acid 

I.  Microchemical  Colorimetric  Method. — Benedict  and  Hitchcock 
Modification  of  the  Folin-Macallmn-Denis  Procedure. — The  principle 
of  the  method  depends  upon  the  fact,  first  noted  by  FoHn  and  Macallum^ 
and  further  investigated  by  Folin  and  Denis, ^  that  uric  acid  gives,  with 
phosphotungstic  acid  and  alkali,  a  deep  blue  color  the  depth  of  which  is 
proportional  to  the  amount  of  uric  acid  present.  Since  certain  other 
substances  present  in  urine  produce  a  similar  blue  color  with  the  phos- 
photungstic acid,  it  is  necessary  to  separate  the  uric  acid  from  them. 
This  is  accomplished  by  precipitation  as  the  silver  salt.  The  silver 
urate  is  subsequently  dissolved  and  treated  with  the  uric  acid  reagent. 

Benedict  and  Hitchcock'*  have  examined  the  method  of  Folin  and 
Denis  and  have  suggested  a  number  of  important  modifications. 

Procedure. — Measure  such  an  amount  of  urine  as  will  contain  from  0.7 
to  1.3  mg.  of  uric  acid  (2  to  4  c.c.  is  usually  the  correct  amount)  into  a  centrifuge 
tube,  dilute  with  water  to  about  5  c.c,  and  add  15  to  20  drops  of  an  ammoniacal 
silver  magnesiiun  solution.^  Mix  the  contents  of  the  tube  with  a  small  stirring 
rod  and  centrifuge  the  tube  for  one  or  two  minutes.  Pour  off  the  supernatant 
liquid,  as  completely  as  possible,  by  inverting  the  tube,  allowing  it  to  drain  a 
moment,  and  then  touching  the  inside  of  the  lip  of  the  tube  with  a  towel  or  piece 
of  filter  paper.  Add  to  the  residue  in  the  tube  two  drops  of  a  5  per  cent  solution 
of  potassium  cyanide  to  dissolve  the  silver  urate,  stir  the  mixture  thoroughly 
with  a  thin  rod,  for  half  a  minute,  add  a  few  drops  (0.5  to  i.o  c.c.)  of  water,  and 
stir  again. ^    Two  c.c.  of  the  uric  acid  reagent^  are  added  and  the  mixture  stirred 

^  Morris:  Jour.  Biol.  Chetn.,  21,  201,  1915. 

2  Folin  and  Macallum:  /.  Biol.  Chem.,  13,  363,  1912. 

^  Folin  and  Denis:  J.  Biol.  Client.,  14,  95,  1913;  ibid.,  13,  469,  1913. 

"•  Benedict  and  Hitchcock:  /.  Biol.  Chem.,  20,  619,  1915;  Benedict:  ibid.,  20,  629,  1915. 

*This  solution  has  the  following  composition: 

3  per  cent  silver  lactate  solution 70  c.c. 

M  agnesia  mixture 30  c.c. 

Concentrated  ammonium  hydroxide  solution 100  c.c. 

'  At  this  point  perfectly  clear  solutions  are  obtained  with  pure  uric  acid  solutions  in  phos- 
phate mixture  or  in  pyridin.  With  urines  some  magnesium  ammonium  phosphateisprecipi- 
tated  with  the  uric  acid,  which  does  not  dissolve  in  the  cyanide.  After  adding  the  two 
subsequent  reagents,  however,  a  perfectly  clear  solution  is  obtained. 

'  Preparation  of  the  Uric  Acid  Reagent. — Place  100  grams  of  sodium  tungstate,  80  c.c. 
of  85  per  cent  phosphoric  acid,  and  750  c.c.  of  distilled  water  in  a  liter  flask.  Boil  the  mix- 
ture with  a  reflux  condenser  for  two  hours,  cool  and  dilute  to  i  liter,  filtering  if  necessary. 


URINE  5 1 1 

again,  after  which  add  lo  c.c.  of  20  per  cent  sodium  carbonate  solution/  transfer 
quantitatively  to  a  50  c.c.  flask,  and  at  the  end  of  about  one-half  minute,  dilute 
to  mark.  Compare  this  solution  in  the  Duboscq  colorimeter  ipage.  4861  with  a 
simultaneously  prepared  solution  obtained  by  treating  5  c.c.  of  the  standard  uric 
acid  solution,-  contained  in  a  50  c.c.  flask,  with  2  drops  of  the  potassium  cyanide 
solution,  2  c.c.  of  the  uric  acid  reagent,  10  c.c.  of  20  per  cent  sodium  carbonate  solu- 
tion, and  diluting  to  the  mark  at  the  end  of  about  one-half  minute.  The  stand- 
ard solution  is  best  set  at  a  height  of  15  mm.  in  the  colorimeter. 

Calculation. — The  reading  of  the  standard  divided  by  the  reading  of  the  urine 
gives  the  number  of  miUigrams  of  uric  acid  in  the  amount  of  sample  taken. 

Interpretation. — For  adults  on  a  mixed  diet  the  average  excretion  of 
uric  acid  is  about  0.7  gram.  It  arises  from  the  purines  of  ingested  food 
(exogenous  uric  acid)  and  from  purines  derived  from  the  body  tissues 
by  disintegration  of  nuclein  material  (endogenous  uric  acidj.  Exog- 
enous uric  acid  depending  entirely  upon  the  diet  is  greatly  increased 
by  the  ingestion  of  purine-rich  foods  (meat,  liver,  sweetbreads,  etc.)  and 
reduced  to  a  very  low  level  on  purine-free  foods,  e.g.,  milk,  eggs,  etc. 
(see  Chapter  XXVII).  Endogenous  uric  acid  is  influenced  by  exercise 
and  by  the  diet  (protein  foods  particularly  giving  rise  to  increases). 
It  appears  to  be  partly  the  result  of  gastro-intestinal  secretory  activity. 
On  a  purine-free  diet  the  average  excretion  is  0.1-0.5  gram.  On  a  high 
purine  diet  the  uric  acid  output  may  be  2  grams  per  day. 

In  gout  the  uric  acid  content  of  the  urine  is  low  preceding  an  attack 
and  increases  during  the  attack,  this  fall  and  rise  being  more  or  less 
characteristic.  The  excretion  rises  after  atophan  administration  ap- 
parently due  to  increased  kidney  activity.  In  leukemia  the  excretion 
is  extremely  high  due  to  nuclear  destruction.  The  uric  acid  content 
of  the  urine  is  of  importance  in  relation  to  the  formation  of  uric  acid 
calculi.  The  administration  of  alkali  carbonates  and  citrates  by  de- 
creasing the  acidity  of  the  urine  increases  its  solvent  power  for  uric 
acid,  and  decreases  the  liability  of  the  formation  of  this  type  of  calculus. 

3.  Folin-Shailer  Method.^ — Principle. — The  uric  acid  is  precipitated 
as  ammonium  urate  by  the  addition  of  ammonia,  the  precipitate  filtered 
off,  washed  and  titrated  with  potassium  permanganate.     A  preliminary 

1  Sodium  Carbonate  Solution. — Dissolve  200  grams  of  anhydrous  sodium  carbonate  in 
warm  water  and  make  up  to  i  liter. 

-Standard  Uric  Acid  Solution. — The  solution  of  uric  acid  in  phosphate  solution  is  very 
readily  prepared,  does  not  need  to  be  standardized,  and  appears  to  keep  indefinitely.  It  is 
prepared  in  the  following  manner.  Dissolve  9  grams  of  pure  crystallized  disodium  hydro- 
gen phosphate,  together  with  i  gram  of  crystallized  sodium  dihydrogen  phosphate,  in  200 
to  300  c.c.  of  hot  water,  and  tilter  if  the  solution  is  not  perfectly  clear.  Make  this 
filtrate  up  to  about  500  c.c.  with  hot  wafer,  and  pour  this  hot  or  warm  (and  perfectly  clear) 
solution  upon  exactly  200  mg.  of  pure  uric  acid  suspended  in  a  few  cubic  centimeters  of 
water  in  a  liter  volumetric  flask.  Agitate  the  mixture  for  a  few  minutes  until  the  uric  acid 
completely  dissolves.  Cool,  add  exaclly  1.4  c.c.  of  glacial  acetic  acid,  dilute  lo  the  mark, 
and  mix.  Add  about  5  c.c.  of  chloroform  to  prevent  the  growth  of  bacteria  or  moulds  in  the 
solution.     Five  c.c.  of  this  solution  contains  exactly  i  mg.  of  uric  acid. 

'  Folin  and  Shaffer:  Zcil.  physiol.  Chcm.,  32,  552,  1901. 


512  PHYSIOLOGICAL    CHEMISTRY 

treatment  with  an  ammonium  sulphate-uranium  acetate  solution  is  for 
the  purpose  of  removing  interfering  organic  substances.  The  method 
gives  accurate  results. 

Procedure. — Introduce  loo  c.c.^  of  urine  into  an  Erlenmeyer  flask,  add 
25  c.c.  of  the  Folin-Shafifer  reagent-  and  after  shaking  the  flask  to  thoroughly 
mix  the  fluids  allow  the  mixtiire  to  stand,"  with  or  without  further  stirring,  until 
the  precipitate  has  settled  (^5-10  minutes).  Filter,  transfer  100  c.c.  of  the  filtrate 
to  a  200  c.c.  Erlenmeyer  flask,  add  5  c.c.  of  concentrated  ammonimn  hydroxide 
and  allow  the  mixture  to  stand  for  24  hours.  Transfer  the  precipitated  ammo- 
niiun  urate  quantitatively  to  a  filter  paper,  ^  using  10  per  cent  ammoniimi  sulphate 
to  remove  the  final  traces  of  the  urate  from  the  flask.  Wash  the  precipitate 
approximately  free  from  chlorides  by  means  of  10  per  cent  ammonium  sulphate 
solution,^  remove  the  paper  from  the  fvmnel,  open  it,  and  by  means  of  hot  water 
rinse  the  precipitate  back  through  the  fvuinel  into  the  flask  in  which  the  urate 
was  originally  precipitated.  The  volmne  of  fluid  at  this  point  should  be  about 
100  c.c.  Cool  the  solution  to  room  temperature,  add  15  c.c.  of  concentrated 
sulphuric  acid  and  titrate  at  once  with  N/20  potassium  permanganate,  K2Mn208, 
solution.  The  first  tinge  of  pink  color  which  extends  throughout  the  fluid  after 
the  addition  of  two  drops  of  the  permanganate  solution,  while  stirring  with  a 
glass  rod,  should  be  taken  as  the  end-reaction.  Take  the  burette  reading  and 
compute  the  percentage  of  uric  acid  present  in  the  urine  under  examination. 

Calculation. — Each  cubic  centimeter  of  N/20  potassium  permanganate  solu- 
tion is  equivalent  to  3.75  mg.  (0.00375  gram)  of  uric  acid.  The  100  c.c.  from 
which  the  ammonivmi  urate  was  precipitated  is  equivalent  to  only  four -fifths 
of  the  100  c.c.  of  urine  originally  taken;  therefore  we  must  take  five-fourths  of 
the  burette  reading  in  order  to  ascertain  the  nvmiber  of  cubic  centimeters  of  the 
permanganate  solution  required  to  titrate  100  c.c.  of  the  original  urine  to  the  correct 
end  point.  If  y  represents  the  nvmiber  of  cubic  centimeters  of  the  permanganate 
solution  required,  we  may  make  the  following  calculation : 

yXo. 00375  =  weight  of  uric  acid  in  100  c.c.  of  urine. 

Because  of  the  solubility  of  the  ammonitun  urate  a  correction  of  3  mg.  should 
be  added  to  the  final  result. 

Calculate  the  quantity  of  uric  acid  in  the  24-hour  urine  specimen. 

4.  Heintz  Method. — This  is  a  very  simple  method  and  was  the  first  one  in 
general  use  for  the  quantitative  determination  of  uric  acid.  It  is  less  accurate  than 
the  metBods  just  described.  The  procedure  is  as  follows:  Place  100  c.c.  of  filtered 
urine  in  a  beaker,  add  5  c.c.  of  concentrated  hydrochloric  acid,  stir  the  fluid  thor- 
oughly, and  stand  it  away  in  a  cool  place  for  24  hours.  Filter  off  the  uric  acid 
crystals  upon  a  washed,  dried  and  weighed  filter  paper  and  wash  them  with  cold 
distilled  water,  a  few  cubic  centimeters  at  a  time,  until  the  chlorides  are  removed. 

^  It  is  preferable  to  use  more  than  100  c.c.  of  urine  if  the  fluid  has  a  specific  gravity  less 
than  1 .020. 

^  The  Folin-Shaflfer  reagent  consists  of  500  grams  cf  ammonium  sulphate,  5  grams  of 
uranium  acetate  and  60  c.c.  of  10  per  cent  acetic  acid  in  650  c.c.  of  distilled  water. 

'  The  mixture  should  not  be  allowed  to  stand  for  too  long  a  time  at  this  point,  since  uric 
acid  may  be  lost  through  precipitation. 

*  The  Schleicher  and  Schiill  hardened  papers  or  the  Baker  and  Adamson  washed,  ashless 
variety  are  very  satisfactory  for  this  purpose. 

*  This  washing  may  be  conveniently  done  by  decantation  if  desired,  thus  retaining  the 
major  portion  of  the  precipitate  in  the  flask. 


URINE 


513 


Purine  Bases 


Now  wash,  in  turn,  with  alcohol  and  with  ether  and  finally  dry  the  paper  and 
tals  to  constant  weight  at  iio°C.  In  the  process  of  washing  the  uric  acid  free 
chlorides  an  error  is  introduced,  since  every  cubic  centimeter  of 
water  so  used  dissolves  0.00004  gram  of  uric  acid.^  For  this  rea- 
son a  correction  is  necessary.  It  has  been  suggested  that  the  pig- 
ment of  the  crystals  is  equivalent  in  weight  to  the  amount  of  uric 
acid  dissolved  by  the  first  30  c.c.  of  water,  and  this  factor  should  be 
taken  into  account  in  the  computation  of  the  percentage  of  uric 
acid. 

Calculation. — Since  100  c.c.  of  urine  was  used  the  corrected  , 
weight  of  the  uric  acid  crystals,  in  grams,  will  express  the  percentage  \ 
of  uric  acid  present.  1 

5.  Kriiger-Schmidt  Method. — Kriiger  and  Schmidt  have  de-     j 
vised  a  method  for  the  combined  determination  of  uric  acid  and     { 
the  other  purine  bodies  of  urine.      This  procedure  is  described  under     ! 
Purine  Bases,  below.     A  modification  of  this  method  by  Hunter 
is  also  given. 

6.  Ruhemann's  Uricometer  Method. — Principle. — When  iodine 
solution  is  added  to  urine  an  amount  of  iodine  is  absorbed  roughly 
proportional  to  the  amount  of  the  uric  acid  present.  A  special  grad- 
uated tube  is  required  and  carbon  disulphide  is  used  to  indicate  the 
presence  of  excess  iodine. 

Procedure. — Fill  the  tube  (see  Fig.  165)  to  the  lowest  mark  S 
with  carbon  disulphide  (the  bottom  of  the  meniscvis  should  rest 
on  the  line).  Then  add  iodine  solution  (1.5  grams  iodine,  1.5 
grams  potassium  iodide,  15  grams  absolute  alcohol,  and  185  grams 
distilled  water)  to  the  mark  J.  Add  urine  to  mark  2.45  (2.6  c.c). 
Insert  the  glass  stopper  and  shake.  The  carbon  disulphide  will 
show  the  dark  brown  color  of  iodine.  Add  more  urine  gradually 
with  continued  shaking  untU  only  a  violet-pink  color  remains  in  the 
disulphide  after  shaking.  Then  add  drop  by  drop  with  vigorous 
shaking  until  the  disulphide  becomes  colorless.  Read  on  the  scale 
the  amount  of  uric  acid  in  parts  per  thousand  of  urine. 

When  the  uric  acid  content  of  the  urine  is  low  this  method 
gives  reasonably  accurate  results  from  the  cUnical  standpoint. - 
At  a  level  of  0.3  gram  per  day  the  probable  error  will  not  exceed 
10-15  per  cent.  Above  i  gram  no  limit  can  safely  be  placed  on 
the  degree  of  error  in  pathological  cases  especially  where  albumin, 
diacetic  acid,  purine  bases  and  certain  drugs  are  likely  to  be  present. 
The  results  by  this  method  are  ordinarily  high. 


crys- 
from 


ox  in* 

o.in 

0.178 
0.181 
O.IM 
O.tST 
0.190 
0.198 
0.196 
0.199 
0.202 
0.20» 
0.208 
0.211 
0315 
0.219 
0.221 
0.225 
0.228 
fl.23l 
fl.235 
0.238 
0.M2 
0.245 
0.249 

o.nt 

026 
0.28 


Fig.  165. — 
Ruhemann's 
Uricometer. 


I.  Kriiger  and  Schmidt's  Method. — Principle. — This 
method  serves  for   the  determination  of  both  uric  acid 
and  the  purine  bases.     The  principle  involved  is  the  precipitation  of 
both  the  uric  acid  and  the  purine  bases  in  combination  with  copper 

1  His  and  Paul:  Zeit.  physiol.  Chcrn.,  31,  i,  1900. 

*  Bradley  and  Bunta:  Jour.  Am.  Med.  Assn.,  Jan.  4,  1913. 

33 


514  PHYSIOLOGICAL    CHEMISTRY 

oxide  and  the  subsequent  decomposition  of  this  precipitate  by  means 
of  sodium  sulphide.  The  uric  acid  is  then  precipitated  by  means  of 
hydrochloric  acid  and  the  purine  bases  are  separated  from  the  filtrate 
in  the  form  of  their  copper  or  silver  compounds.  The  nitrogen  content 
of  the  precipitates  of  uric  acid  and  purine  bases  is  then  determined 
by  means  of  the  Kjeldahl  method  (see  page  483)  and  the  correspond- 
ing values  for  uric  acid  and  purine  bases  calculated. 

Procedure. — To  400  c.c.  of  albumin-free  urine^  in  a  liter  flask-,  add  24 
grams  of  sodixun  acetate,  40  c.c.  of  a  solution  of  sodium  bisulphite^  and  heat 
the  mixtiire  to  boiling.  Add  40-80  c.c.^  of  a  10  per  cent  solution  of  copper  sul- 
phate and  maintain  the  temperature  of  the  mixture  at  the  boiling-point  for  at 
least  three  minutes.  Filter  off  the  flocculent  precipitate,  wash  it  with  hot 
water  until  the  wash  water  is  colorless,  and  return  the  washed  precipitate  to 
the  flask  by  pimcturing  the  tip  of  the  filter  paper  and  washing  the  precipitate 
through  by  means  of  hot  water.  Add  water  imtil  the  volume  in  the  flask  is 
approximately  200  c.c,  heat  the  mixture  to  boiUng  and  decompose  the  precipi- 
tate of  copper  oxide  by  the  addition  of  30  c.c.  of  sodium  sulphide  solution.* 
After  decomposition  is  complete,  the  mixture  should  be  acidified  with  acetic 
acid  and  heated  to  boiling  imtil  the  separating  sulphur  collects  in  a  mass. 
Filter  the  hot  flmd  by  means  of  a  filter-pump,  wash  with  hot  water,  add  10 
c.c.  of  10  per  cent  hydrochloric  acid  and  evaporate  the  filtrate  in  a  porcelain 
dish  imtil  the  total  volume  has  been  reduced  to  about  10  c.c.  Permit  this 
residue  to  stand  about  two  hours  to  allow  for  the  separation  of  the  uric  acid, 
leaving  the  purine  bases  in  solution.  Filter  off  the  precipitate  of  uric  acid, 
using  a  small  filter  paper,  and  wash  the  uric  acid,  with  water  made  acid  with 
sulphuric  acid,  until  the  total  volume  of  the  original  filtrate  and  the  wash  water 
aggregates  75  c.c.  Determine  the  nitrogen  content  of  the  precipitate  by  means 
of  the  Kjeldahl  method  (see  page  483),  and  calculate  the  uric  acid  equivalent.^ 

Render  the  filtrate  from  the  uric  acid  crystals  alkaline  with  sodium  hydroxide, 
add  acetic  acid  until  faintly  acid  and  heat  to  7o°C.  Now  add  i  c.c.  of  a  10  per 
cent  solution  of  acetic  acid  and  10  c.c.  of  a  suspension  of  manganese  dioxide^ 
to  oxidize  the  traces  of  uric  acid  which  remain  in  the  solution.  Agitate  the  mix- 
ture for  one  minute,  add  10  c.c.  of  the  sodium  bisulphite  solution^  and  5  c.c. 
of  a  10  per  cent  solution  of  copper  sulphate  and  heat  the  mixture  to  boiling  for 

'  If  albumin  is  present,  the  urine  should  be  heated  to  boiling,  acidified  with  acetic  acid, 
and  filtered. 

2  The  total  volume  of  urine  for  the  24  hours  should  be  suflaciently  diluted  with  water  to 
make  the  total  volume  of  the  solution  1600-2000  c.c. 

*  A  solution  containing  50  grams  of  Kahlbaum's  commercial  sodium  bisulphite  in  100 
c.c.  of  water. 

*  The  exact  amount  depending  upon  the  content  of  the  purine  bases. 

'This  is  made  by  saturating  a  i  per  cent  solution  of  sodium  hydroxide  with  hydrogen 
sulphide  gas  and  adding  an  equal  volume  of  i  per  cent  sodium  hydroxide. 

Ordinarily  the  addition  of  30  c.c.  of  this  solution  is  sufficient,  but  the  presence  of  an 
excess  of  sulphide  should  be  proven  by  adding  a  drop  of  lead  acetate  to  a  drop  of  the  solution. 
Under  these  conditions  a  dark  brown  color  will  snow  the  presence  of  an  excess  of  sodium 
sulphide. 

'This  may  be  done  by  multiplying  the  nitrogen  value  by  three  and  adding 3 J^  mg. 
to  the  product  as  a  correction  for  the  uric  acid  remaining  in  solution  in  the  75  c.c. 

^  Made  by  heating  a  0.5  per  cent  solution  of  potassium  permanganate  with  a  little  alco- 
hol until  it  is  decolorized. 

*  To  dissolve  the  excess  of  manganese  dioxide. 


URINE 


:)i:> 


three  minutes.  Filter  off  the  precipitate,  wash  it  with  hot  water,  and  determine 
its  nitrogen  content  by  means  of  the  Kjeldahl  method  (see  page  483).  Inas- 
much as  the  composition  and  proportion  of  the  purine  bases  present  in  urine  is 
variable,  no  factor  can  be  appUed.  The  result  as  regards  these  bases  must 
therefore  be  expressed  in  terms  of  nitrogen. 

Benedict  and  Saiki'  report  cases  in  which  the  total  purine  nitrogen  by  this 
method  was  less  than  the  uric-acid  nitrogen  as  determined  by  the  Fohn-Shaffer 
method.  The  inaccuracy  was  found  to  he  in  the  Kxiiger  and  Schmidt  method. 
To  obviate  this  they  advise  the  addition  of  20  c.c.  of  glacial  acetic  acid  for  each 
300  c.c.  of  urine  employed,  the  acid  being  added  before  the  first  precipitation. 

Interpretation. — The  amount  of  purine  bases  excreted  by  a  normal 
man  is  small  and  variable.  Values  from  16-60  mg.  have  been  found. 
The  purine  base  nitrogen  is  of  course  only  a  fraction  of  this.  The 
amount  excreted  is  influenced  by  the  diet  somewhat  in  the  same  way 
as  is  the  excretion  of  uric  acid  being  also  increased  in  disorders  asso- 
ciated with  increased  uric  acid  excretion  such  as  leukemia.  The  purine 
bases  form  a  higher  percentage  of  the  total  purine  excretion  in  the  case 
of  the  monkey,  sheep,  and  goat  than  in  the  case  of  man. 

2.  Hunter  and  Givens'  Modification  of  Kriiger-Schmidt  Method.  ^ — 
Principle. — The  Kriiger-Schmidt  process  is  combined  ^\dth  the  micro- 
chemical  colorimetric  method  for  uric  acid  (see  page  510). 

Procedure. — The  first  copper-purine  precipitate  as  obtained  in  the  Kriiger- 
Schmidt  procedvu'e  is  suspended  in  about  200  c.c.  of  water,  to  which  there  is 
added  about  i  c.c.  of  concentrated  hydrochloric  acid.  The  mixture  is  vigorously 
boiled,  whereupon  the  whole  or  greater  part  of  the  precipitate  goes  into  solution. 
Removal  of  the  copper  is  effected  by  treatment  with  hydrogen  sulphide  in  the  heat, 
and  excess  of  the  sulphide  is  completely  expelled  by  renewed  boihng.  Filtration 
under  suction,  and  thorough  washing  of  flask  and  filter  result  in  a  filtrate  which 
is  perfectly  clear  and  nearly  colorless.  This  is  concentrated  if  necessary,  and 
made  up  to  a  convenient  volume  which  must  of  course  be  sufficiently  large  to 
retain,  when  cool,  the  uric  acid  in  solution.  Of  this  an  aUquot  part  is  utihzed 
directly  for  the  colorimetric  determination  of  uric  acid.  In  the  remainder  the 
residual  luic  acid  is  destroyed  and  bases  determined  according  to  the  regular 
Kriiger-Schmidt  procedure.  This  modification  is  recommended  particularly 
where  the  amount  of  uric  acid  present  is  minute. 

3.  Welker's  Modification  of  the  Methods  of  Amstein  and  of  Salkowski.^ — 
Principle. — The  phosphates  are  removed  by  treatment  with  magnesia  mixture. 
The  purine  bases  and  uric  acid  are  then  thrown  down  as  their  silver  salts  and  the 
nitrogen  content  of  this  precipitate  determined. 

Procedure. — Four  hundred  c.c.  of  urine,  free  from  protein,  are  treated  with 
100  c.c.  of  magnesia  mixture  and  600  c.c.  of  water.  This  is  then  filtered  and  of  the 
clear  filtrate  a  measured  quantity  (600-800  c.c.  )is  treated  with  an  excess  (10  c.c.) 
of  a  3  per  cent  silver  nitrate  solution.  Concentrated  ammonium  hydroxide  is 
added  in  small  quantities,  with  stirring,  until  all  the  chlorides  have  dissolved. 

1  Benedict  and  Saiki:  Jour.  Biol.  Chem.,  7,  27,  1909. 
^Hunter  and  Givens:  Jour.  Biol.  Chem.,  17,37,  1914. 
^  Dittman  and  Welker:  Xew  York  Med.  Jour.,  May-June,  1909. 


5l6  PHYSIOLOGICAL   CHEMISTRY 

Allow  the  flocculent  precipitate  of  the  silver  purine  compounds  to  settle  to  the 
bottom,  then  pass  the  supernatant  liquid  through  the  filter  before  disturbing  the 
precipitate.  Finally  transfer  the  precipitate  quantitativelj'  to  the  paper  which 
must  be  of  known  nitrogen  content.  The  precipitate  is  washed  with  dilute  (i  per 
cent)  ammonium  hydroxide.  The  paper  with  the  precipitate  is  then  transferred 
to  a  Kjeldahl  flask  and  about  loo  c.c.  of  water  and  a  small  quantity  (about  o.i 
gram)  of  magnesium  oxide  are  added.  The  water  is  then  boiled  until  all  the  am- 
monia has  been  driven  off.     Test  the  steam  with  litmus  paper. 

The  material  in  the  flask  is  then  digested  by  means  of  the  usual  Kjeldahl  method 
(see  page  483).  The  digestion  must  be  watched  carefully  at  the  time  the  sulphuric 
acid  reaches  sufficient  concentration  to  affect  the  filter  paper,  inasmuch  as  the  SO2 
produced  causes  considerable  frothing.  The  total  nitrogen  (purine  base,  uric  acid 
and  filter-paper  nitrogen)  is  now  determined  in  the  usual  way  (see  Kjeldahl  Method, 
page  483).  This  result  minus  the  uric  acid  and  filter-paper  nitrogen  will  give  the 
figure  for  the  purine-base  nitrogen. 

4.  Salkowski's  Method. — Principle. — The  purin  bases  and  uric  acid  are  pre- 
cipitated as  silver-magnesium  salts,  these  compounds  decomposed  with  hydrogen 
sulphide,  and  the  uric  acid  precipitated  by  means  of  sulphuric  acid.  The  purin 
bases  in  the  filtrate  are  again  precipitated  as  their  silver  salts  and  the  silver  content 
■of  the  precipitate  determined  after  ignition  by  titration  with  thiocyanate. 

Procedure. — Place  400-600  c.c.  of  protein-free  urine  in  a  beaker.  Introduce 
into  another  beaker  30-50  c.c.  of  an  ammoniacal  silver  nitrate  solution^  wih  30-50 
•c.c.  of  magnesia  mixture,^  add  some  ammonium  hydroxide  and  if  necessary  some 
ammonium  chloride  to  clear  the  solution.  Now  add  this  solution  to  the  urine, 
stirring  continually  with  a  glass  rod,  and  allow  the  mixture  to  stand  for  one-half 
hour.  Collect  the  precipitate  on  a  filter  paper,  wash  it  with  dilute  ammonium 
hydroxide,  and  finally  wash  it  back  into  the  original  beaker.  Suspend  the  precipi- 
tate in  600-800  c.c.  of  water,  add  a  few  drops  of  hydrochloric  acid  and  decompose 
it  by  means  of  hydrogen  sulphide.  Now  heat  the  solution  to  boiling,  filter  while 
hot  and  evaporate  the  filtrate  to  dryness  on  a  water-bath.  Extract  the  residue 
with  20-30  c.c.  of  hot  3  per  cent  sulphuric  acid  and  allow  the  extract  to  stand  24 
hours.  Filter  off  the  uric  acid,  wash  it,  make  the  filtrate  ammoniacal  and  precipitate 
the  purine  bases  again  with  silver  nitrate.  Collect  this  precipitate  on  a  small-sized 
chlorine-free  filter  paper,  wash,  dry,  and  incinerate  it  in  the  usual  manner.  Now 
dissolve  the  ash  in  nitric  acid  and  titrate  with  ammonium  thiocyanate  according  to 
the  Volhard-Arnold  method  (see  page  556).  Calculate  the  content  of  purine  bases 
in  the  urine  examined,  bearing  in  mind  that  in  an  equal  mixture  of  the  silver  salts 
of  the  purine  bases,  such  as  we  have  here,  one  part  of  silver  corresponds  to  0.277 
gram  of  nitrogen  or  to  0.7381  gram  of  the  bases. 

For  interpretation  see  page  515. 

Purine  Nitrogen 

Hall's  Purinometer.' — By  means  of  the  instrument  shown  in  Fig.  166,  urine 
may  be  examined  for  total  purine  nitrogen,  i.e.,  nitrogen  in  the  form  of  purine  bases, 

^  Prepared  by  dissolving  26  grams  of  silver  nitrate  in  about  500  c.c.  of  water,  adding 
enough  ammonium  hydroxide  to  redissolve  the  precipitate  which  forms  upon  the  first  addi- 
tion of  the  ammonia  and  making  the  balance  of  the  mixture  up  to  i  liter  with  water. 

^  Directions  for  preparation  may  be  found  on  p.  602. 

2  Hall:  The  Purine  Bodies,  Philadelphia,  1904. 


URINE 


517 


urates  and  uric  acid.  The  method  does  not  give  an  absolutely  accurate  measure 
of  the  purine  values.  It  is,  however,  of  considerable  service  clinically.  The 
principle  of  the  method  is  the  preliminary  precipitation  of 
the  phosphates  present  followed  by  the  precipitation  of  the 
purine  bodies  in  the  form  of  their  silver  compounds  by  means 
of  an  ammoniacal  silver  nitrate  solution.  The  volume  of  this 
silver  precipitate  is  then  determined  and  its  nitrogen  value 
interpolated  by  means  of  a  table  of  equivalent  values. 

Procedure. — Collect  the  24-hour  urine  and  mix  it 
thoroughly.  Take  100  c.c.  of  the  urine  and  if  albumin  is 
present  make  slightly  acid  with  acetic  acid  and  boil  and  filter. 
Close  the  stopcock  of  the  instrument  and  introduce  90  c.c. 
of  urine  and  20  c.c.  of  a  modified  magnesia  mixture.*  Turn 
the  stopcock  and  permit  the  precipitated  phosphates  to  pass 
into  the  lower  chamber  of  the  instrument.  After  an  interval 
of  ten  minutes  has  elapsed  the  stopcock  should  be  closed 
and  sufficient  ammoniacal  silver  nitrate  solution^  added  to 
make  the  total  volume  in  the  upper  chamber  100  c.c.  The 
precipitate  of  the  silver  compounds  of  the  purine  bodies 
should  be  pale  yellow,  Anj^  silver  chloride  present  may  be 
brought  into  solution  in  the  strong  ammoniacal  solution  by 
the  repeated  inversion  of  the  purinometer.  In  case  the 
chloride  does  not  dissolve  it  should  be  brought  into  solution 
by  the  addition  of  further  ammonium  hydroxide.  Place  the 
purinometer  in  a  dark  room  for  24  hours  and  at  the  end  of 
this  time  read  the  volume  of  the  purine  precipitate.  Inter- 
polate the  value  in  terms  of  purine  nitrogen  by  means  of  the  following  table: 


Fig.      166. — H.a.ll's 
Purinometer. 


Precipitate,  Purine  nitrogen, 

c.c.  per  cent 

(grams  in  100  c.c.) 

4 0.007S 

5 0.0097 

6 0.0117 

7 0.0136 

8 0.0156 

9 0.0175 

10 0.0185 

II 0.0195 

12 O. 0205 

13 0.0218 

14 0.0225 

15 0.0234 

16 0.0249 

17 0.0  2t)0 

18 0.0265 

19 0.0270 

^This  is  prepared  as  follows:  Dissolve  10  grams  of  magnesium  chloride  in  about  75  c.c. 
of  water  and  add  to  grams  of  ammonium  chloride.  Introduce  100  c.c.  of  concentrated 
ammonium  hydro.\ide  into  this  mixture.  If  a  precipitate  forms  add  ammonium  hydroxide 
until  a  clear  solution  is  obtained.  ]Make  the  volume  200  c.c.  by  means  of  water  and  add  10 
grams  of  purified  talcum. 

-This  solution  has  the  following  formula: 

Silver  nitrate i  gram. 

Ammonium  hydroxide  (sp.  gr.  0.90) 100  c.c. 

Talcum 5  grams. 

Distilled  water. .  .  too  c.c. 


5l8  PHYSIOLOGICAL    CHEMISTRY 

Precipitate,  Purine  nitrogen, 

c.c.  per  cent 

(grams  in  loo  c.c.) 

20 0.0275 

21 0.0283 

22 0.0286 

23 o . 0299 

24 0,0312 

25 0.0325 

Calculation. — Multiply  the  purine  nitrogen  percentage  by  the  total  volume  of 
urine  and  divide  by  100  to  obtain  the  total  purine  nitrogen  value.  For  example, 
if  the  precipitate  was  found  to  be  12  c.c.  and  the  total  volume  of  the  24-hour  urine 
was  1300  c.c.  the  calculation  would  be  as  follows: 

12  c.c.  =  0.0205  per  cent  purine  nitrogen. 
0.0205  X  13.0  =  0.2665  gram  purine  nitrogen. 

Interpretation. — The  endogenous  purine  nitrogen  excretion  (on  purine-free 
diet)  averages  about  0.15  gram  per  day,  though  variations  are  considerable.  As 
uric  acid  makes  up  about  nine-tenths  of  this  the  amount  varies  much  as  uric  acid 
varies.  The  exogenous  purine  nitrogen  excretion  may  be  largely  influenced  by  the 
methyl  purines  of  tea,  coffee,  and  cocoa  which  are  excreted  unchanged,  as  well 
as  by  the  purine  bases  which  are  normally  oxidized  to  uric  acid  (see  Uric  Acid, 
page  511). 

Allantoin 

I.  Method  of  Wiechowski-Handovsky.^ — Principle. — The  urine  is  precipitated 
with  phosphotungstic  acid  and  lead  acetate  and  in  the  presence  of  chlorides  with 
silver  acetate.  The  heavy  metals  are  removed  with  hydrogen  sulphide.  The  allan- 
toin is  then  precipitated  as  a  mercuric  compound  and  the  amount  of  mercury  and 
hence  of  allantoin  in  the  precipitate  determined  by  titration  with  ammonium 
thiocyanate.  This  method,  though  rather  tedious,  is  probably  the  most  accurate 
method  for  the  determination  of  allantoin. 

Procedure.— The.  urine  is  diluted  to  about  i  per  cent  urea.  As  rabbit  urine 
contains  in  the  day's  output  about  2-4  grams  of  urea,  and  that  of  other  herbivora 
usually  forms  about  a  4  per  cent  urea  solution,  it  is  usually  desirable  to  dilute  3-4 
times.  A  greater  dilution  is  not  desirable.  The  urine  is  treated  with  i  per  cent  of 
sulphuric  acid  and  about  3  c.c.  of  acetic  acid  for  each  day's  volume.  Test  a  small 
quantity  of  the  urine  to  determine  the  amount  of  50  per  cent  phosphotungstic  acid 
required  to  completely  precipitate  it.  The  bulk  of  the  urine  is  then  treated  on  this 
basis  with  sufficient  solid  phosphotungstic  acid  to  precipitate  it  completely.  Stir 
to  dissolve  the  acid  and  allow  to  stand  for  several  hours.  Filter  with  suction,  first 
lining  the  filter  with  infusorial  earth  by  rubbing  up  a  little  of  the  substance  with  some 
of  the  urine  mixture  and  filtering  with  suction.  To  some  ordinary  lead  oxide  in  a 
mortar  add  a  small  amount  of  the  filtrate  and  stir  until  it  becomes  warm,  then  add 
the  rest  of  the  filtrate  and  stir,  adding  more  oxide  if  necessary  untU  the  solution 
reacts  alkaline  due  to  the  formation  of  basic  lead  acetate. 

Filter  again.     The  filtrate  should  give  no  precipitate  with  basic  lead  acetate. 

A  measured  volume  of  the  filtrate  is  then  treated  with  measured  volumes  of  acetic 

acid  and  silver  nitrate  solution  to  completely  precipitate  any  chlorides  present. 

Filter  again  preferably  through  infusorial  earth.     Pass  hydrogen  sulphide  through 

^  Handovsky:  Zeit.  physinl.  Chem.,  90,  211,  1914. 

VViechowski:  Neubauer-Huppert:  Analyse  des  Hams,  Wiesbaden,  1913,  p.  1076. 


URINE  519 

the  filtrate  until  the  heavy  metals  are  completely  precipitated.  Filter  again.  Pass 
a  current  of  air  through  the  filtrate  to  remove  all  hydrogen  sulphide  (test  with  lead 
acetate  paper).  Neutralize  this  final  filtrate  with  calcium  carbonate  and  remove 
the  carbon  dioxide  with  a  current  of  air. 

The  neutral  filtrate  is  then  treated  with  an  amount  of  allantoin  reageni  in  excess 
of  that  required  to  precipitate  the  allantoin  as  indicated  by  a  preliminary  test. 
(The  allantoin  reagent  is  a  solution  containing  5  per  cent  mercuric  acetate  and  20 
per  cent  of  sodium  acetate.     The  reagent  when  used  must  be  clear.) 

Allow  to  stand  for  half  an  hour  (not  too  long)  and  then  filter.  A  measured 
amount  of  the  filtrate  is  taken  and  treated  with  about  10  c.c.  of  iron-ammonium  alum 
and  the  red  solution  decolorized  with  dilute  sulphuric  acid.  (The  solution  is  not 
completely  decolorized  but  retains  a  faint  greenish  tint.)  Any  calcium  sulphate 
precipitate  formed  at  this  point  may  be  disregarded.  Titrate  with  N/io  ammonium 
thiocyanate  solution  to  a  yellow  color,  which  increases  in  intensity  on  the  addition 
of  1-2  drops  more  of  solution.  The  titration  should  not  be  carried  out  in  artifi- 
cial light  and  some  practice  is  required  to  get  the  exact  end  point.  The  thiocya- 
nate solution  should  be  standardized  occasionally  against  standard  silver  nitrate 
solution. 

Calculation. — One  c.c.  of  X/io  NH4SCN  solution  corresponds  to  0.00436  gram 
of  allantoin.  The  limit  of  error  of  the  method  is  about  5  mg.  for  the  daily  output 
of  allantoin.  In  the  calculation  of  course  all  losses  through  filtration,  etc.,  must 
be  considered. 

For  the  considerable  modifications  required  in  carrying  out  the  determination 
on  human  urine  with  its  very  low  content  of  allantoin  see  the  section  by  Wiechowski 
in  Neubauer-Huppert.^ 

Interpretation. — Allantoin  may  be  found  in  very  small  amounts  in  human  urine 
(5-15  mg.  per  day),  appearing  to  be  mainly,  though  not  entirely,  exogenous  in  origin. 
It  forms,  however,  the  principal  end-product  of  the  purine  metabolism  of  practically 
all  mammals  other  than  man  and  the  anthropoid  apes.  Thus  over  90  per  cent  of 
the  purine-allantoin  nitrogen  excretion  of  the  dog,  the  cow,  and  the  pig  occurs  in 
this  form.  In  these  animals  its  origin  is  from  exogenous  and  endogenous  purines, 
and  its  excretion  is  influenced  by  much  the  same  factors  as  is  that  of  uric  acid  in 
man. 

2.  Determination  by  Difference.— ^Method  of  Plimmer  and  Skelton.- — Allan- 
toin is  transformed  into  urea  and  determined  as  such  in  the  acid-magnesium  chloride 
method  of  Folin  for  urea  in  urine  (page  495).  Urease,  however,  has  no  effect  upon 
allantoin.  Therefore,  determine  the  urea  -|-  allantoin  of  the  urine  according  to  Fo- 
lin's  procedure  (see  Urea),  and  also  determine  the  true  urea  according  to  the 
urease  method  (see  Urea).  The  difference  between  the  results  thus  obtained  rep- 
resents allantoin.  If  sugar  is  present  it  must  be  removed  before  applying  Folin's 
procedure. 

Hippuric  Acid 

I.  Method  of  Folin  and  Flanders.^— Pr/;/677>/f. — The  hippuric  acid 
is  hydrolyzod  to  benzoic  acid  in  alkaline  sohition  and  then  the  solution 
is  boiled  with  strong  nitric  acid  to  remove  pigments  and  emulsifying 

^  Wiechowski:  Neubauer-Huppert;  Analyse  dcs  Hams,  Wiesbaden,  191,^,  p.  1076. 
'^Plimmer  and  Skelton:  Bioclt.  J.,  8,  70  and  641,  1914. 
^  Folin  and  Flanders:  Jottr.  Biol.  Chein  ,  11,  257,  191 2. 


520  PHYSIOLOGICAL   CHEMISTRY 

substances.     The  benzoic  acid  is  extracted  with  chloroform  and  ti- 
trated with  sodium  ethylate. 

Procedtire. — Measure  loo  c.c.  of  urine  into  a  porcelain  evaporating  dish  by 
means  of  a  pipette.  Add  lo  c.c.  of  5  per  cent  NaOH  and  evaporate  to  dryness 
on  the  steam-bath.  Transfer  the  residue  to  a  500  c.c.  Kjeldahl  flask  by  means  of 
25  c.c.  of  water  and  25  c.c.  of  concentrated  HNO3.  Add  0.2  gram  of  copper 
nitrate,  a  couple  of  pebbles  or  glass  pearls  and  boil  very  gently  for  four  and  one- 
half  hours  over  a  micro-burner.  Fit  the  necks  of  the  flasks  with  condensers  of 
the  Hopkins  type  made  from  large  test-tubes  fitted  with  two-hole  rubber  stoppers, 
the  inlet  tubes  extending  near  the  bottom  of  the  test-tubes  while  the  outlet  tube 
is  shorter.  These  condensers  should  fit  rather  loosely.  A  good  current  of  water 
flowing  through  the  condensers  prevents  loss  of  benzoic  acid  or  change  in  con- 
centration of  the  nitric  acid. 

After  cooling,  rinse  the  condensers  down  with  25  c.c.  of  water  and  transfer  the 
contents  of  the  flask  to  a  500  c.c.  separatory  funnel,  with  the  aid  of  25  c.c.  more 
of  water.  The  total  voliune  of  the  solution  is  now  100  c.c.  Add  to  the  solution 
sufl5cient  ammoniimi  sulphate  to  just  saturate  it  (about  55  grams).  Make  four 
extractions  with  freely  washed  chloroform,  using  50,  35,  25,  and  25  c.c.  portions. 
The  first  two  portions  may  be  used  to  further  rinse  out  the  Kjeldahl  flask. 

Collect  the  successive  portions  of  chloroform  in  another  separatory  funnel. 
Add  to  the  combined  extracts  100  c.c.  of  a  saturated  solution  of  pure  sodiimi  chlo- 
ride, to  each  Uter  of  which  has  been  added  0.5  c.c.  of  concentrated  HCl.  Shake 
well,  draw  the  chloroform  into  a  dry  500  c.c.  Erlenmeyer  flask  and  titrate  with  N/io 
sodium  alcoholate,'  using  4  or  5  drops  of  phenolphthalein  as  an  indicator.  The 
first  distinct  end  point  should  be  taken,  although  it  may  fade  on  standing  a  short 
time. 

Calculation. — Multiply  the  mmiber  of  cubic  centimeters  of  alcoholate  used  by 
the  factor  for  hippuric  acid  as  determined  by  standardization  to  obtain  the 
amount  of  hippuric  acid  in  the  100  c.c.  of  urine  used.  One  c.c.  of  exactly  N/io 
sodiimi  alcoholate  is  equivalent  to  0.0179  gram  of  hippuric  acid.  Calculate  the 
daily  output  of  hippuric  acid  from  the  24-hour  volimie. 

Interpretation. — The  average  excretion  of  hippuric  acid  by  a  normal 
adult  man  is  about  0.7  gram  per  day.  The  amount  is  increased  by 
the  ingestion  of  benzoic  acid  or  fruits  containing  it  (plums,  prunes, 
cranberries).  It  arises  in  part  apparently  from  putrefaction  products 
formed  in  the  intestine.  In  herbivora  it  is  often  the  most  abundant 
nitrogenous  constituent  of  the  urine. 

2.  DaMn's  Methods.- — Preliminary  Procedure. — Place  150  c.c.  (or  more)  of  the 
urine  under  examination  in  a  porcelain  evaporating  dish  and  evaporate  almost  to 
dryness  upon  a  water-bath.     Add  about  i  gram  of  sodium  dihydrogen  phosphate, 

'  The  sodium  alcoholate  is  made  by  dissolving  2.3  grams  of  cleaned  metallic  sodium  in  i 
liter  of  absolute  alcohol.  It  is  advisable  that  it  be  slightly  weaker  rather  than  stronger 
than  tenth-normal.  It  may  be  standardized  against  pure  benzoic  acid  in  washed  chloro- 
form. It  may  also  be  standardized  against  N/io  HCl  provided  the  alcoholate  solution 
contains  not  more  than  traces  of  carbonate. 

^  Private  communication  to  the  author  from  Dr.  H.  D.  Dakin. 


URINE  5  2  I 

about  25  grams  of  gypsum  (CaS04,  2H2O)  a^d  rub  up  with  a  pestle  and  stir  with  a 
spatula  until  a  uniform  mixture  results.  Dry  the  powder  thus  produced  in  a  water- 
oven  for  about  two  hours,  at  the  end  of  which  period  it  should  be  rubbed  up  a 
second  time,  to  remove  lumps,  and  transferred  to  a  Schleicher  and  Schiill  "extrac- 
tion shell"  and  extracted  in  a  Soxhlet  apparatus  in  the  usual  way  (see  page  326J. 
The  extraction  medium  is  ethyl  acetate  and  the  flask  containing  the  acetate  should 
be  strongly  heated  over  a  sand-bath^  for  about  two  hours.  The  ethyl  acetate 
extract  is  now  transferred  to  a  separatory  funnel,  and  the  original  flask  rinsed  with 
sufficient  fresh  ethyl  acetate  to  make  the  total  volume  in  the  separatory  funnel- 
about  100  c.c.  Wash  the  ethyl  acetate  solution /?f  times  with  a  saturated  solution 
of  sodium  chloride,  using  8  c.c.  of  the  sodium  chloride  solution  at  each  extraction, 
shaking  vigorously  and  removing  the  sodium  chloride  extract  in  each  case  before 
adding  fresh  sodium  chloride  solution.  The  sodium  chloride  removes  the  urea  com- 
pletely and  the  hippuric  acid  is  then  determined  in  the  urea-free  solution  by  the 
following  volumetric  or  gravimetric  procedure: 

1.  Volumetric  Determination. — Transfer  the  urea-free  ethyl  acetate  solution, 
prepared  as  described  above,  to  a  Kjeldahl  flask,  add  about  25  c.c.  of  water,  a  small 
piece  of  pumice  stone  to  prevent  bumping,  attach  a  condenser  and  distil  off  the 
acetate'  over  a  free  flame.  After  practically  all  of  the  ethyl  acetate  has  been  dis- 
tilled off,  the  nitrogen  in  the  remaining  solution  should  be  determined  by  means  of 
the  Kjeldahl  method  (sec  page  483). 

The  main  source  of  error  in  this  method  is  the  fact  that  any  nitrogen  present 
in  the  form  of  phenaceturic  acid  or  indole  acetic  acid  is  determined  as  hippuric  acid 
nitrogen.     The  error  from  this  source  is,  however,  usually  trifling. 

Calculation. — Calculate  as  usual  for  nitrogen  determinations,   remembering 
that  I  c.c.  o/N/io  sulphuric  acid  is  equivalent  to  0.0179  gro.ni  hippuric  acid. 
Interpretation. — See  page  520. 

2.  Gravi^netric  Determination. — The  urea-free  ethyl  acetate  solution  contained 
in  the  separatory  funnel,  after  washing  with  sodium  chloride  solution,  as  described 
under  Preliminary  Procedure,  page  520,  is  washed  with  5  c.c.  of  distilled  water  to 
remove  the  major  portion  of  the  sodium  chloride.  Transfer  the  solution  from  the 
separatory  funnel  to  a  round-bottomed  flask  and  subject  it  to  a  steam  distillation 
in  the  usual  way.  A  slow  current  of  steam  should  be  used  while  the  ethyl  acetate 
is  being  distilled  off  and  later  a  more  rapid  current  may  be  employed.  The  dis- 
tillation should  be  continued  for  20  minutes.  Now  add  about  o.  i  gram  of  charcoal 
to  the  aqueous  solution  which  is  heated  to  boiling  and  filtered  hot.  Evaporate  the 
solution  in  a  weighed  Jena  glass  dish  on  a  water-bath  until  the  volume  of  the  solu- 
tion is  reduced  to  about  3  c.c.  Stand  the  dish  in  a  warm  place  until  evaporation 
is  complete  and  a  crystalline  residue  remains.  Wash  the  residue,  in  turn,  with  2  c.c. 
of  dry  ether  and  i  c.c.  of  water,  dry  it  in  an  air-bath  at  ioo°C.  and  weigh.  If  it  is 
so  desired  the  residue  may  be  recrystallized  from  a  little  hot  walcr  and  the  melt- 
ing-point determined.  Pure  hippuric  acid  melts  at  i87°C.  Contamination  with 
phenaceturic  acid  may  be  detected  both  by  the  melting-point  and  the  microscopical 
characteristics. 

*  A  water-bath  cannot  be  substituted  inasmuch  as  the  resultant  extraction  would  be 
too  slow. 

-This  ethyl  acetate  solution  contains  hippuric  acid,  urea,  and  other  substances. 

^  The  ethyl  acetate  after  scjiaration  from  the  watery  layer  of  the  distillate  may  be  dried 
over  calcium  chloride  and  used  again.  > 


522  PHYSIOLOGICAL    CHEMISTRY 

Glucose 

I.  Benedict's  Method.^- — Principle.- — Benedict's  reagent  for  the  esti- 
mation of  reducing  sugars  contains  potassium  thiocyanate  as  well  as 
copper  sulphate,  and  in  the  presence  of  the  former  a  white  precipitate 
of  cuprous  thiocyanate  is  formed  on  reduction  instead  of  the  usual  red 
precipitate  of  cuprous  oxide.  The  small  amount  of  potassium  ferro- 
cyanide  also  aids  in  keeping  cuprous  oxide  in  solution.  As  the  pre- 
cipitate formed  is  white  the  loss  of  all  blue  tint  in  the  solution,  indicating 
complete  reduction  of  the  copper,  is  readily  observed.  The  alkali 
used  is  sodium  carbonate,  which  has  the  advantage  over  the  hydroxides 
in  that  there  is  less  danger  of  destruction  of  small  amounts  of  sugar. 
The  solution  also  has  the  great  advantage  of  being  stable  for  an  in- 
definite length  of  time.  The  method  is  recommended  for  simplicity 
and  accuracy. 

Procedure. — The  urine,  lo  c.c.  of  which  should  be  diluted  with  water  to  loo 
c.c.  (unless  the  sugar  content  is  believed  to  be  low,  when  it  may  be  used  undiluted), 
is  povu-ed  into  a  50  c.c.  burette  up  to  the  zero  mark.  Twenty-five  c.c.  of  the 
reagent-  are  measured  with  a  pipette  into  a  porcelain  evaporation  dish  (25-30  cm. 
in  diameter),  10  to  20  grams  of  crystallized  sodimn  carbonate  (or  one-half  the 
weight  of  the  anhydrous  salt)  are  added,  together  with  a  small  quantity  of  pow- 
dered pumice  stone  or  talcum,  and  the  mixture  heated  to  boiling  over  a  free 
flame  until  the  carbonate  has  entirely  dissolved.  The  diluted  urine  is  now  run  in 
from  the  burette,  rather  rapidly,  until  a  chalk-white  precipitate  forms  and  the 
blue  color  of  the  mixture  begins  to  lessen  perceptibly,  after  which  the  solution 
from  the  burette  must  be  nm  in  a  few  drops  at  a  time,  until  the  disappearance  of 
the  last  trace  of  blue  color,  which  marks  the  end  point.  The  solution  must  be 
kept  vigorously  boiling  throughout  the  entire  titration.  If  the  mixture  becomes 
too  concentrated  during  the  process,  water  may  be  added  from  time  to  time  to 
replace  the  volume  lost  by  evaporation. 

Calculation. — The  calculation  of  the  percentage  of  sugar  in  the  original  sample 
of  urine  is  very  simple.  The  25  c.c.  of  copper  solution  are  reduced  by  exactly  50 
mg.  of  glucose.  Therefore  the  volume  run  out  of  the  burette  to  effect  the  reduc- 
tion contained  50  mg.  of  the  sugar.    When  the  luine  is  diluted  i :  10,  as  in  the 

^Benedict:  Jour.  Am.  Med.  Ass'n,  57,  1193,  igii. 

^  Copper  sulphate  (crystallized)  18.0  grams. 

Sodium  carbonate  (crystallized,  one-half  the  weight  of  the 

the  anhydrous  salt  may  be  used) 200. o  grams. 

Sodium  or  potassium  citrate 200.0  grams. 

Potassium  thiocyanate 125.0  grams. 

Potassium  ferrocyanide  (5  per  cent  solution) 5.0  c.c. 

Distilled  water  to  make  a  total  volume  of 1000.  o  c.c. 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate  and  thiocyanate  in  enough  water  to 
make  about  800  c.c.  of  the  mixture  and  filter  if  necessary.  Dissolve  the  copper  sulphate 
separately  in  about  100  c.c.  of  water  and  pour  the  solution  slowly  into  the  other  liquid,  with 
constant  stirring.  Add  the  ferrocyanide  solution,  cool  and  dilute  to  exactly  i  liter.  Of  the 
various  constituents,  the  copper  salt  only  need  be  weighed  with  exactness.  Twenty-five 
c.c.  of  the  reagent  are  reduced  by  50  mg.  of  glucose. 


URINE 


0-0 


usual  titration  of  diabetic  urines,  the  formula  for  calculating  the  per  cent  of  the 

sugar  is  the  following: 

0.050 

X 1000  =  per  cent  in  original  sample,  wherein  X 

is  the  number  of  cubic  centimeters  of  the  diluted  urine  required  to  reduce  25  c.c. 
of  the  copper  solution. 

In  the  use  of  this  method  chloroform  must  not  be  present  during  the  titration. 
If  used  as  a  preservative  in  the  urine  it  may  be  removed  by  boiUng  a  sample  for 
a  few  minutes,  and  then  diluting  to  its  original  volume. 

Interpretation. — Sugar  in  the  urine  in  amounts  sufficient  to  be  de- 
tected by  the  commonly  employed  qualitative  tests  indicates  a  patho- 
logical condition,  unless  very  large  amounts  of  sugar  have  been  ingested 
just  previously,  in  which  case  the  condition  is  spoken  of  as  an  alimentary 
glycosuria.  Persistent  glycosuria  thus  indicates  diabetes  mellitus,  a 
disorder  in  which  the  amount  of  sugar  may  rise  as  high  as  10  per  cent 
and  averages  3-5  per  cent.  The  volume  of  urine  excreted  per  day  is 
usually  also  large  and  the  absolute  sugar  excretion  may  thus  be  very 
great  (100  grams  of  glucose  per  day  are  not  uncommon) .  The  quantita- 
tive methods  for  the  estimation  of  sugar  in  urine  enable  us  to  deter- 
mine the  severity  of  the  disorder  as  well  as  to  follow  its  course  under 
treatment,  etc. 

2.  Fehling's  Method.— Principle. — Diluted  urine  is  run  into  a 
measured  amount  of  Fehling's  solution  at  the  boiling-point  until  all 
of  the  copper  it  contains  is  reduced  as  indicated  by  the  loss  of  blue  color. 
This  method  has  several  disadvantages  over  Benedict's  method.  The 
end  point  is  difficult  to  determine  and  the  mixed  solution  is  unstable. 
It  gives  less  accurate  results. 

Procedure. — Place  10  c.c.  of  the  urine  under  examination  in  a  100  c.c.  volu- 
metric flask  and  make  the  volume  up  to  100  c.c.  with  distilled  water.  (If  the 
urine  contains  less  than  0.5  per  cent  of  sugar  it  may  be  used  without  dilution.  A 
concentration  of  about  0.5  per  cent  is  the  most  satisfactory  for  this  titration.") 
Thoroughly  mix  this  diluted  urine  by  pouring  it  into  a  beaker  and  stirring  with 
a  glass  rod,  then  transfer  a  portion  of  it  to  a  burette  which  is  properly  supported 
in  a  clamp. 

Now  place  10  c.c.  of  Fehling's  solution^  in  a  small  beaker,  dilute  it  with  approxi- 
mately 40  c.c.  of  distilled  water,  heat  to  boiUng,  and  observe  whether  decomposi- 
tion of  the  FehUng's  solution  itself  has  occurred  as  indicated  by  the  production  of 
a  turbidity.  If  such  turbidity  is  produced  the  Fehling's  solution  is  unfit  for  use. 
Clamp  the  burette  containing  the  dilute  urine  immediately  over  the  beaker  and 
carefully  allow  from  0.5-1  c.c.  of  the  diluted  urine  to  flow  into  the  boihng  Fehl- 
ing's solution.  Bring  the  solution  to  the  boiling-point  after  each  addition  of 
urine  and  continue  running  the  urine  from  the  burette,  0.5-1  c.c.  at  a  time,  as  in- 
dicated, until  the  Fehling's  solution  is  completely  reduced,  i.e.,  until  all  the  cupric 

^  Directions  for  the  preparation  of  Fehling's  solution  are  given  in  a  note  at  the  bottom 
of  p.  26. 


524  PHYSIOLOGICAL    CHEMISTRY 

oxide  in  solution  has  been  precipitated  as  cuprous  oxide.  This  point  will  be 
indicated  by  the  absolute  disappearance  of  all  blue  color.  When  this  end  point 
is  reached  note  the  number  of  cubic  centimeters  of  diluted  urine  used  in  the  proc- 
ess and  calculate  the  percentage  of  dextrose  present,  in  the  sample  of  urine 
analyzed,  according  to  the  method  given  below. 

This  is  a  satisfactory  method,  the  main  objection  to  its  use  being  the 
uncertainty  attending  the  determination  of  the  end-reaction,  i.e.,  the  difficulty 
with  which  the  exact  point  where  the  blue  color  finally  disappears  is  noted.  Sev- 
eral means  of  accurately  fixing  this  point  have  been  suggested,  but  they  are 
practically  all  open  to  objection.  As  good  a  "check"  as  any,  perhaps,  is  to  filter 
a  few  drops  of  the  solution  through  a  double  paper,  after  the  blue  color  has 
apparently  disappeared,  acidify  the  filtrate  with  acetic  acid  and  add  potassium 
ferrocyanide.  If  the  copper  of  the  Fehling's  solution  has  been  completely 
reduced,  there  will  be  no  color  reaction,  whereas  the  production  of  a  brown  color 
indicates  the  presence  of  unreduced  copper.  Harrison  has  recently  suggested 
the  following  procedure  to  determine  the  exact  end  point :  To  about  i  c.c.  of  a 
starch  iodide  solution^  in  a  test-tube  add  2-3  drops  of  acetic  acid  and  introduce 
into  the  acidified  mixture  1-2  drops  of  the  solution  to  be  tested.  Unreduced 
copper  will  be  indicated  by  the  production  of  a  purpUsh-red  or  blue  color  due  to 
the  liberation  of  iodine. 

It  is  ordinarily  customary  to  make  at  least  three  determinations  by  Fehling's 
method  before  coming  to  a  final  conclusion  regarding  the  sugar  content  of  the 
urine  under  examination. 

Calculation. — Ten  c.c.  of  Fehhng's  solution  is  completely  reduced  by  0.05 
gram  of  dextrose.-  If  y  represents  the  nmnber  of  cubic  centimeters  of  undiluted 
urine  (obtained  by  dividing  the  burette  reading  by  10)  necessary  to  reduce  the 
10  c.c.  of  Fehling's  solution,  we  have  the  following  proportion. 

y : 0.05 : :  100 : x  (percentage  of  dextrose). 

Interpretation. — See  page  523. 

3.  Bang's  Method.^ — Principle.- — The  solution  to  be  tested  is  boiled 
with  alkaline  cupric  chloride  solution  containing  thiocyanate  and  potas- 
sium chloride.  The  cupric  salt  under  these  conditions  is  reduced  to 
the  cuprous  form  without  any  precipitation  taking  place.  The  re- 
duced copper  is  titrated  with  standard  iodine  solution  using  starch  as 
an  indicator.     The  titration  reaction  is  as  follows: 

CuCl  +  I  +  K0CO3  =  CUCO3  +  KCl  +  KI. 

Procedure. — A  100  c.c,  Jena  flask  with  a  straight  neck  the  flange  of  which  has 
been  cut  off  is  used  for  the  boiUng  procedure.     Over  the  neck  of  the  flask  place 

^  The  starch-iodide  solution  may  be  prepared  as  follows:  Mix  o.i  gram  of  starch  with 
cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into  75-100  c.c.  of  boiling 
water,  stirring  continuously.  Cool  the  starch  paste,  add  20-25  grams  of  potassium  iodide 
and  dilute  the  mixture  to  250  c.c.  This  solution  deteriorates  upon  standing,  and  therefore 
must  be  freshly  prepared  as  needed. 

^  The  values  for  certain  other  sugars  are  as  follows: 

Lactose 0.0676  gram. 

Maltose o. 074     gram. 

Invert  sugar o  o475  gram. 

^  Bang:  Biochem.  Zeit.,  49,  i,  1913. 


URINE 


3^3 


a  piece  of  tight  fitting  rubber  tubing  sufficiently  long  (about  2  inches),  so  that  it 
may  be  provided  with  a  pinch  cock  or  clamp  to  shut  ofif  the  contents  of  the  flask 
from  the  outside  air  (see  Fig.  81,  page  281  j. 

Introduce  into  the  flask  o.i  to  2.0  c.c.  (or  more)  of  the  sugar  solution  contain- 
ing not  more  than  10  mg.  of  glucose.  Then  add  55  c.c.  of  the  diluted  copper 
solution.'  Heat  over  an  asbestos  gauze  with  a  flame  standardized  to  bring  the 
solution  to  the  boiUng-point  in  from  3  12-3  3  4  minutes.  Boil  for  exactly  3 
minutes,  being  prepared  to  close  the  flask  with  the  pinch-cock  at  the  end  of  the 
3  minutes.  Remove  from  the  flame  and  at  once  cool  under  the  tap  to  room  tem- 
perature. Remove  the  rubber  tubing,  add  to  the  contents  of  the  flask  121 
c.c.  of  the  starch  solution  (i  gram  of  soluble  starch  in  100  c.c.  of  saturated  KCl 
solution,  which  keeps  indefinitely).  Titrate  with  the  standard  iodine  solution' 
run  in  from  an  accurate  burette  with  a  glass  stopcock.  When  the  iodine  starch 
color  appears  throughout  the  solution  rotate  gently  and  let  stand  a  few  seconds. 
The  end  point  is  reached  when  the  blue  color  endures  for  10-20  seconds.  It  is 
preferable  to  carry  out  the  titration  in  an  atmosphere  of  carbon  dioxide,  main- 
tained by  means  of  a  deUvery  tube  hung  over  the  side  of  the  flask.  Otherwise 
the  titration  must  be  carried  out  rapidly  to  prevent  reoxidation  by  the  oxygen  of 
the  air. 

Calculation. — Divide  the  number  of  cubic  centimeters  of  N  100  iodine  solu- 
tion used  in  the  titration  by  2.7  to  obtain  the  niunber  of  milligrams  of  glucose  in 
the  amount  of  solution  used. 

This  method  is  suitable  for  urine  analysis.  The  urine  must  however  be  free 
from  albumin  and  as  urine  contains  substances  reacting  slowly  with  iodine  the 
end  point  must  be  taken  when  the  blue  color  persists  for  about  10  seconds  and  any 
slow  decolorization  disregarded. 

Interpretation. — See  page  523. 

4.  Peters'  Method.- — Principle. — The  sugar  solution  is  boiled  with  an  alkaline 
copper  solution  under  rigidly  standardized  conditions  and  after  filtration  the  un- 
reduced copper  is  determined  by  adding  potassium  iodide  and  titrating  the  liber- 
ated iodine  with  standard  thiosulphate  solution. 

Procedure. — .1 .  The  Healing  Power. — It  is  necessary  to  standardize  the  heating 
power  of  the  flame  used  in  the  reduction  process.  A  200  c.c.  Erlenmeycr  flask  of 
Jena  glass  and  of  about  6  cm.  basal  diameter  is  used.  This  bears  a  two-hole  rubber 
stopper,  one  hole  of  which  carries  a  thermometer.     The  lower  end  of  the  thermome- 

^  Copper  solution. — Stock  Solution. — Dissolve  first  160  grams  potassium  bicarbonate, 
100  grams  potassium  carbonate  and  66  grams  of  potassium  chloride  with  about  700  c.c. 
of  distilled  water  in  a  liter  flask.  Pure  salts  must  be  used  in  each  case.  As  the  bicarbonate 
is  diflicultj'  soluble  it  should  be  finely  powdered  and  brought  into  solution  first,  preferably 
with  warming  to  30°C.  The  KCl  is  then  dissolved  and  finally,  with  cooling,  the  carbonate. 
Then  add  100  c.c.  of  a  4.4  per  cent  solution  of  pure  crystalline  CUSO4.5H2O.  Fill  to  the 
mark  with  distilled  water.  ]ML\  without  strong  shaking  and  let  stand  for  24  hours  before 
using. 

Dilute  Copper  Solution. — Dilute  300  c.c.  of  the  stock  solution  to  1000  c.c.  ^lix  with 
only  gentle  shaking.     Allow  to  stand  for  several  hours  before  using. 

Standard  Iodine  Solution. — The  N/ioo  iodine  solution  is  made  by  dilution  of  N/io 
iodine  solution  (see  appendix)  with  boiled  out  distilled  water.  The  solution  is  stable  for 
three  months  if  kept  in  a  dark  bottle.  It  may  also  be  prepared  daily  from  KI  and  KIOj. 
Introduce  into  a  100  c.c.  flask  about  i  c.c.  of  2  per  cent  KIO3.  and2-2.5  grams  of  KI  and 
then  e.xactly  10  c.c.  of  N/io  HCl.  The  HCl  liberates  an  equivalent  amount  of  iodine  (.sul- 
phuric acid  is  less  desirable).     Make  to  100  c.c.  with  distilled  water  and  mix. 

*  Peters:  /.  Am.  Chent.  Soc,  34,  928,  1912;  34,  422,  1912. 


526  PHYSIOLOGICAL   CHEMISTRY 

ter  shovdd  extend  to  about  2  mm.  from  the  bottom  of  the  flask  and  the  35°  mark 
on  the  thermometer  stem  should  be  visible  above  the  stopper.  The  flask  is  placed 
on  an  asbestos  gauze  supported  by  an  adjustable  ring  stand.  A  Bunsen  or  Meker 
burner  is  used  and  is  placed  at  from  3-5  cm.  beneath  the  lower  surface  of  the  asbestos 
gauze.  Use  a  large  flame  and  allow  the  ring  and  gauze  to  become  thoroughly- 
heated.  Then  place  the  flask,  into  which  60  c.c.  of  distilled  water  has  been  intro- 
duced, in  the  center  of  the  gauze  and  observe  the  time  required  for  the  temperature 
in  the  flask  to  rise  from  35  to  95°C.  By  several  trials  the  flame  and  position  of  the 
gauze  are  so  adjusted  that  it  requires  (within  a  few  seconds  either  way)  just  120 
seconds  for  the  temperature  to  rise  from  35-95°C.  This  standard  flame  is  then 
used  in  the  determinations  which  follow. 

B.  The  Reduction  Process. — Into  a  200  c.c.  Jena  Erlenmeyer  flask  fitted  as 
above,  introduce  20  c.c.  of  alkaline  tartrate  solution,^  20  c.c.  of  the  copper  sulphate 
solution^  and  the  sugar  solution  to  be  analyzed  which  should  first  be  made  up  to 
20  c.c.  so  that  the  total  volume  of  the  fluid  in  the  reduction  flask  is  always  60  c.c. 
Place  the  flask  over  the  standard  flame  and  note  when  the  temperature  of  the  mix- 
ture reaches  95°C.  Allow  to  boil  for  exactly  20  seconds  after  the  temperature 
reaches  95°.  Then  the  flask  is  promptly  removed  with  the  stopper  still  in  place 
and  held  under  the  tap  for  a  moment  to  stop  the  reduction  but  not  to  cool  the  mix- 
ture more  than  a  few  degrees  below  the  boiling-point.  Filter  hot  through  a  pre- 
viously prepared  Gooch  crucible  with  a  heavy  asbestos  mat  through  which  suflScient 
of  a  suspension  of  talcum  has  been  filtered  so  that  with  suction,  not  a  stream  but  a 
rapid  succession  of  drops  comes  through  the  filter.  (A  calcium  chloride  tube 
packed  with  glass  wool  and  asbestos  may  also  be  used.)  The  suction  flask  should 
have  a  capacity  of  about  200  c.c.  so  that  the  titration  may  be  carried  out  in  it  di- 
rectly. Wash  the  filter  with  a  fine  stream  of  water  using  not  more  than  15-20  c.c. 
of  water  in  all.  To  the  filtrate  add  4  c.c.  of  concentrated  sulphuric  acid  and  cool 
to  2o°C.  Add  6-7  c.c.  of  a  saturated  solution  of  potassium  iodide.  Titrate  the 
liberated  iodine  with  standard  thiosulphate  solution,^  adding  a  few  drops  of  a  solu- 
tion of  soluble  starch  as  an  indicator  near  the  close  of  the  titration.  The  "spot 
test "  may  be  used  in  determining  the  end  point.  As  long  as  a  drop  of  thiosulphate 
produces  a  perceptible  white  area  in  falling  upon  the  quiet  solution  the  end  point 
has  not  been  reached.  The  chocolate-brown  color  of  the  mixture  changes  to  a 
light  cream  white  as  the  last  necessary  drop  of  thiosulphate  is  added. 

A  blank  should  be  run  in  exactly  the  same  way  but  with  the  omission  of  the 
sugar  solution. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  thiosulphate  re- 
quired for  the  titration  of  unreduced  copper  from  the  number  of  cubic  centimeters 
required  for  the  blank.     This  gives  the  amount  of  thiosulphate  equivalent  to  copper 

^  Copper  Solution. — Dissolve  34,639  grams  of  highest  purity  crystallized  copper  sulphate 
(Kahlbaum's  "zur  Analyse  mit  Garantieschein)  in  water  to  make  500  c.c. 

Alkaline  Tartrate  Solution. — Dissolve  173  grams  of  sodiumpotassium  tartrate  and  125 
grams  of  potassium  hydroxide  in  water  to  make  500  c.c. 

^  N/5  Sodium  Thiosulphate. — Dissolve  about  50  grams  of  ordinary  c.p.  sodium  thio- 
sulphate or  exactly  49.66  grams  of  the  pure,  dry,  recrystalHzed  salt,  in  enough  boiled-out 
distilled  water  to  make  a  liter.  Allow  to  stand  for  several  days.  The  solution  should  be 
standardized  against  the  copper  solution  prepared  as  above.  For  this  purpose  introduce 
20  c.c.  of  the  copper  solution  into  a  200  c.c.  Erlenmeyer  flask,  add  20  c.c.  of  30  per  cent 
acetic  acid  and  40  c.c.  of  water.  Add  about  7  grams  of  a  saturated  solution  of  KI  and 
titrate  with  the  thiosulphate  using  starch  as  an  indicator.  Calculate  the  equivalent  of  i 
c.c.  of  thiosulphate  in  Cu.  One  c.c.  of  the  copper  sulphate  solution  contains  17.647  mg. 
of  Cu.  The  thiosulphate  remains  constant  for  some  months.  It  should  be  kept  in  a  dark 
bottle. 


URINE  527 

reduced.  Multiply  this  result  by  the  value  of  i  c.c.  of  thiosulphate  expressed  as 
Cu,  and  obtain  the  number  of  milligrams  of  copper  reduced.  Then  by  consulting 
the  table  of  values  (below)  determine  the  weight  of  glucose  equivalent  to  this 
amount  of  copper. 

Cole^  has  determined  the  copper  values  for  lactose  using  this  method  exactly 
as  outlined.  He  has  also  suggested  the  following  empirical  formula  which  agrees 
well  with  the  values  derived  from  his  table: 

Mg.  anhydrous  lactose  =  1.25  +  0.77S  X  mg.  Cu. 

Inter pr elation. — See  page  523. 

TABLE  FOR  THE  DETERMINATION  OF  GLUCOSE 
According  to  Peters 


Glucose 

Copper, 
mg. 

Glucose 

ratio 
Cu 

Glucose 

Coi)pcr, 
mg. 

Glucosc 

ratio 
Cu 

I 

1 .2 

0-833 

60 

II5-5 

0.522 

2 

2.8 

0.714 

70 

134-4 

0.522 

5 

8.2 

0.610 

80 

152-9 

0.522 

8 

13-8 

0.580 

90 

171. 0 

0.522 

10 

17-4 

0-575 

100 

191. 6 

0.522 

15 

27.7 

0.542 

no 

208.9 

0.527 

20 

371 

0-539 

120 

228.1 

0.526 

25 

48.1 

0.522 

135 

255-0 

0.529 

30 

57-3 

0.522 

150 

280.8 

0.534 

35 

67.6 

0.522 

165 

306.8 

0.538 

40 

76.2 

0.522 

180 

330-5 

0.545 

45 

86.0 

0.522 

200 

349-6 

0-572 

50 

96.0 

0.  522 

5.  Bertrand's  Method.''  Principle. — The  sugar  is  boiled  with  alkaline  copper 
sulphate  solution  and  the  precipitated  cuprous  oxide  filtered  off,  dissolved  in  an 
acid  solution  of  ferric  sulphate  and  the  resultant  ferrous  salt  titrated  with  potassium 
permanganate.  This  method  of  titrating  cuprous  oxide  may  be  conveniently  used 
where  other  reduction  procedures  such  as  that  of  Peters  or  of  Munson  and  Walker,* 
have  been  employed.  In  this  case  the  tables  corresponding  to  the  particular 
method  and  not  the  Bertrand  tables  must  be  consulted  in  calculating  the  sugar 
equivalent. 

The  following  reactions  are  involved  in  the  Bertrand  titration: 

CuoO  +  Fe2(S04)3  +  H2SO4  =  2CUSO4  +  2FeS04  +  HjO 
loFeSO*  +  2KMn04  +  8H2SO4  =  5Fe2(S04)s  +  K2SO4  +  2MnS04  -f  8H,0. 

Procedure. — Introduce  into  an  Erlenmeyer  flask  of  150  c.c.  capacity,  20  c.c.  of 
the  sugar  solution  (of  a  concentration  of  0.5  per  cent  or  less),  20  c.c.  of  the  copper 

^Cole:  Biochem.  J.,  8,  134,  1914- 

*  Bertrand:  BM.  Soc.  Chivi.  de  France,  35,  1285,  1906. 

'Munson  and  Walker:  Bull.  107,  U.  S.  Dept.  of  Agriculture. 


52S 


PHYSIOLOGICAL    CHEMISTRY 


TABLE  FOR  THE  DETERMINATION  OF  REDUCING  SUGARS 
According  to  Bertrand 


Sugar  in  mg. 

Glucose 
Cu 

Invert  sugar 
Cu 

Galactose 

rcu 

Maltose 
Cu 

Lactose 
Cu 

lO 

20.4 

20.6 

19  3 

II .  2 

14.4 

II 

22.4 

22 

6 

21.2 

12 

3 

15 

8 

12 

24-3 

24 

6 

23.0 

13 

4 

17 

2 

13 

26.3 

26 

5 

24.9 

14 

5 

18 

6 

14 

28.3 

28 

5 

26.7 

15 

6 

20 

0 

15 

30.2 

30 

5 

28.6 

16 

7 

21 

4 

16 

32.2 

32 

5 

30-5 

17 

8 

22 

8 

17 

34-2 

34 

5 

32.3 

18 

9 

24 

2 

18 

36.2 

36 

4 

34-2 

2D 

0 

25 

6 

19 

38.1 

38 

4 

36.0 

21 

I 

27 

0 

20 

40.  r 

40 

4 

37-9 

22 

2 

28 

4 

21 

42.0 

42 

3 

39-7 

23 

3 

29 

8 

22 

43-9 

44 

2 

41.6 

24 

4 

31 

I 

:           ^3 

45-8 

46 

I 

43-4 

25 

5 

32 

5 

24 

47-7 

48 

0 

45-2 

26 

6 

33 

9 

25 

49.6 

49 

8 

47.0 

27 

7 

35 

2 

26 

51-5 

51 

7 

48.9 

28 

9 

36 

6 

27 

53-4 

53 

6 

50.7 

30 

0 

38 

0 

28 

55-3 

55 

5 

52. 5 

31 

I 

39 

4 

29 

57-2 

57 

4 

54-4 

32 

2 

40 

7 

30 

59   I 

59 

3 

56.2 

33 

3 

42 

I 

31 

60.9 

61 

I 

58.0 

34' 

4 

43 

4 

32 

62.8 

63 

0 

59-7 

35 

5 

44 

8 

33 

64.6 

64 

8 

61. s 

36 

5 

46 

I 

34 

66.5 

66 

7 

633 

37 

6 

47 

4 

35 

68.3 

68 

5 

65.0 

38 

7 

48 

7, 

36 

70.1 

70 

3 

66.8 

39 

8 

50 

I 

37 

72.0 

72 

2 

68.6 

40 

9 

51 

4 

38 

73-8 

74 

0 

70.4 

41 

9 

52 

7 

39 

75-5 

75 

9 

72.1 

43 

0 

54 

I 

40 

77-5 

77 

7 

73-9 

44 

I 

55 

4 

•  41 

79-3 

79 

5 

75-6 

45 

2 

56 

7 

42 

81. 1 

81 

2 

77-4 

46 

3 

58 

0 

43 

82.9 

83 

0 

79.1 

47 

4 

59 

3 

44 

84.7 

84 

8 

80.8 

48 

5 

60 

6 

45 

86.4 

86 

5 

82.5 

49 

5 

61 

9 

46 

88.2 

88 

3 

84-3 

50 

6 

63 

3 

47 

90.0 

90 

I 

86.0 

51 

7 

64 

6 

48 

91.8 

91 

9 

87.8 

52 

8 

65 

9 

49 

93-6 

93 

6 

895 

53 

9 

67 

2 

50 

95-4 

95 

4 

91 .2 

55 

0 

68 

5 

51 

97.1 

97 

1 

92.9 

56 

I 

69 

8 

52 

98.9 

98 

8 

94.6 

57 

I 

71 

I 

53 

100.6 

100 

6 

96.3 

58 

2 

72 

4 

54 

102.3 

102 

3 

98.0 

59 

3 

73 

7 

55 

104. 1 

104 

0 

99-7 

60 

3 

74 

9 

i          56 

1 

105.8 

los 

7 

101.5 

61 

4      , 

76 

2 

URINE 


529 


TABLE   FOR   THE    DETERMINATION    OF   REDUCING    SVG ARS.— (Continued) 

According  to  Bertrand 


Glucose 

Invert  sugar 

Galactose 

Maltose 

Lactose 

Sugar  in  mg. 

Cu 

Cu 

Cu 

Cu 

Cu 

S7 

107.6 

107.4 

103.2 

62.5 

77-5 

58 

109.3 

109.  2 

104.9 

63-5 

78.8 

59 

III.  I 

no. 9 

106.6 

64.6 

80.1 

60 

112. 8 

112. 6 

108.3 

65-7 

81.4 

61 

II4-5 

114-3 

no.o 

66.8 

82.7 

62 

116. 2 

115.9 

111.6 

67-9 

839 

63 

117. 9 

117. 6 

II3-3 

68.9 

85.2 

64 

119. 6 

119.  2 

115.0 

70.0 

86.5 

65 

121. 3 

120.9 

116.6 

71.1 

87.7 

66 

123.0 

122.6 

118.3 

72.2 

89  0 

67 

124.7 

124.2 

120.0 

73-3 

90.3 

68 

126.4 

125.9 

121.7 

74-3 

91.6 

69 

128. 1 

127-S 

123.3 

75-4 

92.8 

70 

129.8 

129.2 

125.0 

76.5 

94.1 

71 

131-4 

130.8 

126.6 

77-6 

95.4 

72 

133- 1 

132.4 

128.3 

78.6 

96.6 

73 

134-7 

134.0 

130.0 

79-7 

97-9 

74 

136.3 

135-6 

131.5 

80.8 

99-1 

75 

137-9 

137.2 

133-1 

81.8 

100.4 

76 

139.6 

138.9 

134-8 

82.9 

lOI  .7 

77 

141.  2 

140.5 

136.4 

84.0 

102.9 

78 

142.8 

142. 1 

138.0 

85.1 

104.2 

79 

144-5 

143-7 

139.7 

86.1 

105.4 

80 

146. 1 

145-3 

141.3 

87.2 

106.7 

81 

147-7 

146.9 

142.9 

83.3 

107.9 

82 

149-3 

148. 5 

144.6 

89-4 

109.2 

83 

150.9 

150.0 

146.2 

90.4 

110.4 

84 

152. 5 

151. 6 

147.8 

91-5 

III. 7 

85 

154-0 

153-2 

149.4 

92.6 

112.9 

86 

155-6 

154-8 

151. 1 

93-7 

114. 1 

87 

157-2 

156.4 

152.7 

94-8 

II5-4 

88 

158.8 

157-9 

154.3 

95-3 

116. 6 

89 

160.4 

159-5 

156.0 

96.9 

117.9 

90 

162.0 

161.1 

157.6 

98.0 

119. 1 

91 

163.6 

162.6 

159-2 

99.0 

120.3 

92 

165.2 

164.2 

160.8 

100. 1 

121. 6 

93 

166.7 

165.7 

162.4 

10:.  1 

122.8 

94 

168.3 

167.3 

164.0 

102.2 

124.0 

95 

169.9 

168.8 

165.6 

103.2 

125.2 

96 

171-S 

170.3 

167.2 

104.  2 

126.5 

97 

173-1 

171.9 

168.8 

105.3 

127.7 

98 

174-6 

173-4 

170.4 

106.3 

128.9 

99 

176.  2 

175-0 

172.0 

107.4 

130.2 

100 

177-8 

^  -(''    ' 

173 -6 

108.4 

131    4 

34 


530  PHYSIOLOGICAL    CHEMISTRY 

solution^  and  20  c.c.  of  the  alkaline  tartrate  solution.^  Heat  to  boiling  over  an 
asbestos  gauze  and  boil  gently  for  exactly  three  minutes.  Let  stand  a  moment  that 
the  copper  oxide  may  settle  and  then  filter  with  suction  through  a  Gooch  crucible 
with  a  heavy  asbestos  mat  (or  a  calcium  chloride  tube  with  successive  layers  of 
glass  wool,  coarse  asbestos  and  fine  asbestos  -wool)  into  a  flask  of  about  150  c.c. 
capacity.  Wash  the  residue  in  the  flask  twice  by  decantation  with  a  little  hot 
water  pouring  the  supernatant  liquid  through  the  filter.  Throw  away  the  clear 
filtrate,  rinse  the  flask  and  replace  it.  To  the  flask  containing  the  cuprous  oxide 
add  5-20  c.c.  of  the  acid  ferric  sulphate  solution.^  A  green  solution  containing 
ferrous  sulphate  is  formed.  Pour  through  the  filter  together  with  a  little  more  of 
the  acid  solution  if  necessary  to  completely  dissolve  the  copper  oxide.  Wash  flask 
and  filter  with  a  little  water.  Titrate  the  filtrate  with  standard  potassium  per- 
manganate solution^  to  a  rose  color.  The  procedure  should  be  carried  out  as  rapidly 
as  possible. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  permanganate  used 
by  its  copper  equivalent  as  determined  by  standardization,  and  from  the  table 
(page  528-529)  obtain  the  corresponding  value  for  the  sugar  under  examination. 

Interpretation — See  page  523. 

6.  Fermentation  Method. — Principle. — This  method  consists  in 
the  measurement  of  the  volume  of  carbon  dioxide  evolved  when  the 
dextrose  of  the  urine  undergoes  fermentation  with  yeast.  None  of 
the  various  methods  whose  manipulation  is  based  upon  this  principle 
is  absolutely  accurate.  The  method  in  which  Einhorn's  saccharometer 
(Fig.  3,  page  31)  is  the  apparatus  employed  is  perhaps  as  satisfactory 
as  any  for  clinical  purposes. 

Procedure. — Place  about  15  c.c.  of  urine  in  a  mortar,  add  about  i  gram  of 
yeast  (1/16  of  the  ordinary  cake  of  compressed  yeast)  and  carefully  crush  the 
latter  by  means  of  a  pestle.  Transfer  the  mixture  to  the  saccharometer,  being 
careful  to  note  that  the  graduated  tube  is  completely  filled  and  that  no  air  bubbles 
gather  at  the  top.  Allow  the  apparatus  to  stand  in  a  warm  place  (30°C.)  for  12 
hours  and  observe  the  percentage  of  dextrose  as  indicated  by  the  graduated  scale 
of  the  instrument.  Both  the  percentage  of  dextrose  and  the  number  of  cubic 
centimeters  of  carbon  dioxide  are  indicated  by  the  graduations  on  the  side  of  the 
saccharometer  tube. 

The  fermentation  method  becomes  a  much  more  accurate  procedure 
if  the  saccharometer  of  Lohnstein  is  used.^ 

^  (a)  Copper  Sulphate  Solution. — Forty  grams  of  pure  crystallized  copper  sulphate  are 
dissolved  in  water  to  make  a  liter. 

Q))  Alkaline  Tartrate  Sol uti 071. — Dissolve  200  grams  of  Rochelle  salts  and  150  grams  of 
NaOH  in  water  to  make  1000  c.c. 

(c)  Acid  Ferric  Sulphate  Solution. — Dissolve  50  grams  of  ferric  sulphate  in  about  200  c.c. 
of  water  and  pour  into  this  a  mixture  of  200  c.c.  of  concentrated  sulphuric  acid  diluted 
with  about  400  c.c.  of  water.     Mix  and  make  up  to  1000  c.c. 

{d)  Potassium  Permanganate  Solution. — Dissolve  5  grams  of  potassium  permanganate  in 
water  to  make  1000  c.c.  Standardization. — Dissolve  0.250  gram  of  ammonium  oxalate  in 
50-100  c.c.  of  water,  add  1-2  c.c.  of  concentrated  sulphuric  acid  and  titrate  with  the  per- 
manganate to  a  rose  color.  About  22  c.c.  will  be  required.  Multiplj^  the  number  of  grams 
of  oxalate  used  by  0.895  to  get  the  equivalent  in  Cu  of  the  number  of  cubic  centimeters  of 
permanganate  used.     Calculate  the  Cu  value  of  i  c.c. 

^Lohnstein:  MUnch.  med.  Woch.,  1S99,  1671. 


URINE 


531 


The  availability  of  the  fermentation  procedure  as  a  quantitative 
aid  has  been  appreciably  lowered  through  the  important  findings  of 
Neuberg  and  Associates.^  They  show  that  yeast  has  the  property  of 
splitting  off  carbon  dioxide  from  the  carhoxyl  group  of  amino-  and  other 
aliphatic  acids.  The  active  agent  in  this  "sugar-free  fermentation"  is 
an  enzyme  called  carboxylase.  Inasmuch  as  amino-acids  are  always 
present  in  the  urine,  the  error  from  this  source  is  apparent. 

7.  Polariscopic  Examination. — Before  subjecting  urine  to  a  polariscopic 
examination  the  slightly  acid  fluid  should  be  decolorized  as  thoroughly  as  possible 
by  the  addition  of  a  little  lead  acetate.  The  urine  should  be  well  stirred  and  then 
filtered  through  a  filter  paper  which  has  not  been  previously  moistened.  In  this 
way  a  perfectly  clear  and  almost  colorless  Uquid  is  obtained. 

In  determining  dextrose  by  means  of  the  polariscope  it  should  be  borne  in 
mind  that  this  carbohydrate  is  often  accompanied  by  other  optically  active  sub- 
stances, such  as  proteins,  fructose,  /3-hydroxybutyric  acid,  and  conjugate  gly- 
curonates  which  may  introduce  an  error  into  the  polariscopic  reading ;  the  method 
is,  however,  sufiiciently  accurate  for  practical  piuposes. 

For  directions  as  to  the  manipulation  of  the  polariscope  see  page  31. 

Below  are  given  the  specific  rotations  of  some  physiologically  important  sugars 
as  well  as  of  certain  other  optically  active  substances  the  possible  presence  of 
which  must  be  borne  in  mind  in  determining  glucose  polarimetrically  in  urine. 


Specific  Rotation 


Specific  Rotation 


Glucose 

+  52.49 

Fructose 

-92.25 

Maltose 

+  136.5 

.:(-Hydroxybutyric 

—  24.12 

Isomaltose 

+68.0 

acid. 

Lactose 

+  52.53 

Conjugated   Gly- 

Levorotatory  in 

Pentose  (i-ara- 

0.0 

curonic  Acids. 

varying  degrees. 

binose). 

Protein 


I.  Scherer's  Coagulation  Method.  The  content  of  coagulable  protein 
may  be  accurately  determined  as  follows  :  Place  50  c.c.  of  urine  in  a  small  beaker 
and  raise  the  temperature  of  the  fluid  to  about  40°C.  upon  a  water-bath.  Add 
dilute  acetic  acid,  drop  by  drop,  to  the  warm  urine,  to  precipitate  the  protein  which 
will  separate  in  a  flocculent  form.  Care  should  be  taken  not  to  add  too  much 
acid ;  ordinarily  less  than  20  drops  is  sufiicient.  The  temperature  of  the  water  in 
the  water-bath  should  now  be  raised  to  the  boiUng-point  and  maintained  there 
for  a  few  minutes  in  order  to  insure  the  complete  coagulation  of  the  protein  pres- 
ent.    Now  filter  the  urine-  through  a  previously  washed,  dried,  and  weighed 

1  Xeuberf,'  and  Associates:  Biochcm.  Zeilschr.,  vol.  31  and  36,  iqii. 

-  If  it  is  desired  the  precipitate  may  be  filtered  off  on  an  unweighed  paper,  and  its  nitrogen 
content  determined  by  the  Kjeldahl  method  (see  p.  483).  In  order  to  arrive  at  correct 
figures  for  the  protein  content  it  is  then  simply  necessary  to  multiply  the  total  nitrogen 
content  by  6.25  (see  p.  328).  Correction  should  be  made  for  the  nitrogen  content  of  the 
filter  paper  used  unless  this  factor  is  negligible. 


532 


PHYSIOLOGICAL   CHEMISTRY 


filter  paper,  wash  the  precipitated  protein,  in  turn,  with  hot  water,  95  per  cent 
alcohol,  and  with  ether,  and  dry  the  paper  and  precipitate,  to  constant  weight,  in 
an  air-bath  at  i  io°C.  Subtract  the  weight  of  the  filter  paper  from  the  combined 
weight  of  the  paper  and  precipitate  and  calculate  the  percentage  of  protein  in  the 
urine  specimen. 

Calculation. — To  determine  the  percentage  of  protein  present  in  the  urine 
under  examination,  multiply  the  weight  of  the  precipitate,  ex- 
pressed in  grams,  by  2. 

Interpretation. — The  amount  of  albumin  occurring 
in  the  urine  is  not  necessarily  an  index  of  the  severity 
or  type  of  the  disorder  giving  rise  to  it.  Hence  no  sig- 
nificant figures  can  be  given.  Normal  human  urine 
probably  contains  a  trace  of  albumin  which  is  too  slight 
to  be  detected  or  determined  by  the  usual  procedures. 
The  determination  of  albumin  may  be  of  assistance  in 
following  the  course  of  kidney  disturbances,  but  the  re- 
sults can  be  interpreted  only  in  the  light  of  other  clinical 
findings. 

2.  Esbach's  Method. — This  method  depends  upon  the  pre- 
cipitation of  protein  by  Esbach's  reagent^  and  the  apparatus 
used  in  the  estimation  is  Esbach's  albuminometer  (Fig.  167). 
In  making  a  determination  fill  the  albmninometer  to  the 
point  U  with  urine,  then  introduce  the  reagent  imtil  the 
point  R  is  reached.  Now  stopper  the  tube,  invert  it  slowly 
several  times  in  order  to  insure  the  thorough  mixing  of  the 
fluids,  and  stand  the  tube  aside  for  24  hours.  Creatinine,  resin, 
acids,  etc.,  are  precipitated  in  this  method,  and  for  this  and 
other  reasons  it  is  not  as  accurate  as  the  coagulation  method. 
It  is,  however,  extensively  used  clinically.  According  to  Sahli^ 
the  method  is  "accurate  approximately  to  one  part  per  1000," 
whereas  Pfeiffer^  claims  it  is  not  accurate  for  less  than  one- 
half  or  for  more  than  five  parts  per  1000. 

Calculation. — ^The  graduations  on  the  albuminometer  indi- 
cate grams  of  protein  per  liter  of  urine.  Thus,  if  the  protein 
precipitate  is  level  with  the  figure  3  of  the  graduated  scale,  this 
denotes  that  the  urine  examined  contains  3  grams  of  protein  to 
the  liter.  To  express  the  amotmt  of  protein  in  per  cent  simply  move  the  deci- 
mal point  one  place  to  the  left.  In  the  case  under  consideration  the  urine 
contains  0.3  per  cent  protein. 

Interpretation. — See  above. 

3.  Kwilecki's  Modification  of  Esbach's  Method.^ — Add  10  drops  of  a  10  per 
cent  solution  of  FeCU  to  the  acid  urine  before  introducing  the  Esbach's  reagent. 

^  Esbach's  reagent  is  prepared  by  dissolving  10  grams  of  picric  acid  and  20  grams  of 
citric  acid  in  i  liter  of  water. 

^Sahli:  Lehrbuch  d.  klin.  U titer suchungs-Methoden,  5th  Aufl.,  1909. 
3  Pfeiffer:  Berl.  klin.  Woch.,  49,  114,  191 2. 
*  Kwilecki:  Miinch  med.  Woch.,  56,  p.  1330. 


Fig.  167.— 
Esbach's  Albu- 
minometer. 


URINE 


533 


Wann  the  tube  and  contents  in  a  water-bath  at  72°C.  for  5-6  minutes  and  make 
the  reading. 

4.  Turbidity  Method  of  Folin  and  Denis.' — Principle. — The  albumin  of  the 
urine  is  precipitated  with  sulphosalicylic  acid  and  the  turbidity  produced  com- 
pared with  that  of  a  standard  protein  solution. 

Procedure. — To  about  75  c.c.  of  water  in  each  of  two  100  c.c.  volumetric  flasks 
is  added  5  c.c.  of  a  25  per  cent  solution  of  sulphosalicylic  acid.  To  one  flask  is  then 
added  5  c.c.  of  the  standard  protein  solution  containing  10  mg.  of  albumin  and  to 
the  other  is  added  the  albuminous  urine  i  c.c.  at  a  time  (by  means  of  an  Ostwald 
pipette)  until  the  turbidity  obtained  seems  to  be  reasonably  near  that  of  the 
standard.  The  two  flasks  are  then  filled  up  to  the  mark  with  water,  cautiously 
inverted  a  few  times  to  secure  mixing,  and  are  then  ready  for  the  quantitative 
comparison  just  as  in  colorimetric  work.  The  standard  must  invariably  be  read 
against  itself.  The  standard-  containing  10  mg.  of  protein  is  set  at  20  mm.  The 
unknown  must  not  read  less  than  10  nor  more  than  30. 

Calculation. — Dividing  200  by  the  product  of  the  reading  of  the  unknown  and 
the  number  of  cubic  centimeters  of  urine  taken  gives  the  albumin  in  milligrams  per 
cubic  centimeter  of  urine.  The  albuminous  suspensions  must  not  be  shaken  but 
mixed  very  carefully.  The  method  is  fairly  accurate  and  requires  but  a  few  minutes 
if  a  standard  solution  is  at  hand.  The  method  is  not  applicable  to  urines  deeply 
colored  with  blood  or  bile  but  may  be  used  for  albuminous  fluids  other  than  urine 
if  such  fluids  are  not  highly  pigmented.  It  must  be  borne  in  mind  that  different 
proteins,  as  serum  albumin  and  serum  globulin,  may  give  markedly  different  degrees 
of  turbidity  under  the  same  conditions.^ 

Interpretation. — See  page  532. 

Acetone  and  Acetoacetic  Acid 

I.  Folin-Hart  Method. — Principle. — This  method  as  well  as  the 
two  following  serve  the  purpose  of  determining  the  acetone,  and  aceto- 
acetic acid  together  in  terms  of  acetone.  Acetoacetic  acid  on  heating 
is  decomposed  with  the  formation  of  acetone.  In  this  method  the 
acetone  preformed  and  that  derived  from  the  acetoacetic  acid  are 
carried  over  by  means  of  an  air  current  into  a  solution  of  iodine  in  potas- 
sium hydroxide  (alkaline  hypoiodite  solution).  The  acetone  is  here 
absorbed  and  retained  as  iodoform.  The  excess  iodine  over  that 
necessary  to  combine  with  the  acetone  is'determined  by  titration  with 
sodium  thiosulphate.  The  method  is  simple  and  accurate  if  directions 
are  carefully  followed. 

^  Folin  and  Denis:  Jour.  Biol.  Client.,  iS,  273,  1914. 

-  Standard  Albumin  solution.  Fresh  blood  serum  free  from  hemoglobin  is  used. 
25-35  c.c.  of  the  serum  are  diluted  with  a  15  per  cent  solution  of  chemically  pure  sodium 
chloride  to  about  1500  c.c.  The  solution  is  mixed  and  filtered.  By  means  of  nitrogen 
determinations  the  protein  content  of  the  filtrate  is  determined  (protein  =  XX6. 25)  and 
on  the  basis  of  the  figure  obtained  the  solution  is  diluted  with  15  per  cent  sodium  chloride 
solution  so  that  it  contains  2  mg.  of  protein  per  cubic  centimeter.  It  is  best  to  saturate 
the  albumin  solution  with  chloroform.     The  solution  keeps  for  months. 

'Marshall  and  Banks:  Proceedings  of  the  American  Philosophical  Society,  54,  176, 
1915. 


534  PHYSIOLOGICAL   CHEMISTRY 

Procedure, — ^Introduce  into  a  wide-mouth  bottle  200  c.c.  of  water,  an  accu- 
rately measured  excess  of  N/io  iodine  solution^and  an  excess  of  40  per  cent  potas- 
sium hydroxide.  Prepare  an  aerometer  cylinder  containing  alkaline  hypoiodite 
solution  to  absorb  any  acetone  which  may  be  present  in  the  air  of  the  laboratory, 
and  between  the  cylinder  and  bottle  suspend  a  test-tube  about  2  inches  in  diam- 
ter.  This  large  test-tube  should  contain  20  c.c.  of  the  urine  under  examination, 
ID  drops  or  a  10  per  cent  solution  of  phosphoric  acid,  10  grams  of  sodium  chloride 
and  a  Uttle  petroleum,  and  should  be  raised  sufficiently  high  to  facilitate  the  easy 
application  of  heat  to  its  bottom  portion.  The  connections  on  the  side  of  the  tube 
should  be  provided  with  bulb-tubes  containing  cotton.  When  the  apparatus  is 
arranged  as  described,  it  should  be  connected  with  a  Chapman  pmnp  and  an  air 
current  passed  through  for  twenty-five  minutes.  During  this  period  the  contents 
of  the  test-tube  are  heated  just  to  the  boiling-point  and  after  an  interval  of  five 
minutes  again  heated  in  the  same  manner.  By  this  means  the  diacetic  acid  is 
converted  into  acetone  and  at  the  end  of  the  twenty-five-minute  period  this 
acetone,  as  well  as  the  preformed  acetone,  will  have  been  removed  from  the  urine 
to  the  absorption  bottle  and  there  retained  as  iodoform. 

The  contents  of  the  absorption  bottle  should  now  be  acidified  with  concen- 
trated hydrochloric  acid,-  and  titrated  with  N/io  sodiimi  thiosulphate  and  starch 
as  in  the  Messinger-Huppert  method  (see  below). 

Interpretation. — iNormal  adults  on  a  mixed  diet  excrete  on  the 
average  3-15  mg.  of  combined  acetone  and  acetoacetic  acid  per  day  and 
anything  over  20  mg.  is  usually  pathological.  The  amount  is  con- 
siderably increased  in  fasting  and  on  a  carbohydrate-free  diet  due  to  the 
development  of  acidosis.  In  severe  diabetic  acidosis  values  up  to  6 
grams  per  day  or  even  higher  may  be  noted.  It  is  sometimes  found  in 
large  amounts  in  intoxications  associated  with  pregnancy.  It  may  be 
found  in  increased  amounts  in  the  urine  in  a  great  variety  of  patho- 
logical conditions.  Quantitative  estimation  enables  us  to  follow  the 
course  of  the  acidosis.     Ammonia  excretion  is  also  largely  increased  in 

1  Proceed  as  follows  in  order  to  obtain  a  rough  idea  regarding  the  amount  of  N/io 
iodine  solution  to  be  used:  Introduce  into  a  test-tube  10  c.c.  of  the  urine  under  examina- 
tion and  I  c.c.  of  a  solution  of  ferric  chloride  made  by  dissolving  100  grams  of  ferric  chloride 
in  100  c.c.  of  distilled  water.  After  permitting  the  mixture  to  stand  for  two  minutes, 
compare  the  color  with  that  of  an  equal  volume  of  the  ferric  chloride  solution  in  a  test- 
tube  of  similar  diameter.  If  the  two  solutions  be  of  approximately  the  same  color  intensity, 
20  c.c.  of  the  urine  under  examination  will  yield  sufl&cient  acetone  to  require  nearly  10  c.c. 
of  N/io  iodine  solution.  In  case  the»nuxture  is  darker  in  color  than  is  the  ferric  chloride 
solution,  the  former  should  be  diluted  with  distilled  water  untU  it  is  of  approximately  the 
same  intensity  as  the  ferric  chloride  solution.  From  this  data  the  amount  of  N/io  iodine 
solution  required  may  be  roughly  estimated  by  means  of  the  following  table: 


Urine  c.c. 

Ferric  chloride 

Water 

N/io  Iodine  required  c.c. 

xo 

I 
I 

I 
I 

10 

10 
10 
10 

10 
20 
30 

20 

35 
40 

^  An  excess  of  iodine  is  indicated  by  the  development  of  a  brown  color. 


URINE  535 

these  conditions,  being  used  in  the  neutralization  of  the  excess  acids 
formed  in  the  body.  Usually  about  three-quarters  of  the  combined 
acetone  and  acetoacetic  acid  excretion  is  in  the  form  of  acetoacetic  acid, 
but  the  proportion  is  not  constant. 

Acidosis  is  due  mainly  to  a  disturbance  in  the  metabolism  of  fats. 
The  fatty  acids  are  ordinarily  oxidized  through  hydroxybutyric  acid  to 
acetoacetic  acid  which  is  in  turn  oxidized  through  formic  and  acetic 
acids  to  carbon  dioxide  and  water.  When  fat  catabolism  is  increased 
to  such  an  extent  that  the  body  cannot  bring  about  complete  oxidation 
of  the  products  formed,  a  considerable  portion  of  the  acetoacetic  acid 
instead  of  being  oxidized  in  this  way  is  transformed  into  acetone 
(a  process  taking  place  but  to  a  slight  extent  normally).  This  acetone 
as  well  as  acetoacetic  acid  and  in  more  severe  cases  /3-hydroxy butyric 
acid  will  then  be  eliminated  to  varying  degrees  in  the  urine. 

The  relation  of  the  acetone  bodies  is  indicated  in  the  following 
scheme : 

CH3— CHOH— CH2— COOH  (/3-hydroxybutyric  acid) 

oxidation    I  T  reduction 

CH3— CO— CH2— COOH  (acetoacetic  acid) 

I  loss  of  CO2 

CH3 — CO — CHa  (acetone) 

In  fasting  the  decomposition  of  fat  is  increased  due  to  the  lack  of 
carbohydrate  material  and  acidosis  develops.  The  same  holds  true 
for  a  carbohydrate-free  diet.  Apparently  also  fat  is  much  less  readily 
oxidized  in  the  presence  of  a  carbohydrate  deficiency. 

2.  Messinger-Huppert  Method. — Principle. — This  method  differs  from  the 
preceding  in  that  distillation  of  the  acetone  is  substituted  for  aspiration.  It  is  an 
accurate  method  of  determining  acetone  and  diacetic  acid  together. 

Procedure. — Place  100  c.c.  of  urine  in  a  distillation  flask  and  add  2  c.c.  of  50 
per  cent  acetic  acid.  Connect  the  flask  with  a  condenser,  properly  arrange  a  re- 
ceiver, attach  a  terminal  series  of  bulbs  containing  water,  and  distil  over  about 
nine-tenths  of  the  urine  mixture.  Remove  the  receiver,  attach  another,  and  sub- 
ject the  residual  portion  of  the  mi.xture  to  a  second  distillation.  Test  this  fluid  for 
acetone  and  if  the  presence  of  acetone  is  indicated  add  about  100  c.c.  of  water  to 
the  residue  and  again  distil.  Treat  the  united  acetone  distillates  with  i  c.c.  of 
dilute  (12  per  cent)  sulphuric  acid  and  redistil,  collecting  this  second  distillate  in  a 
glass-stoppered  flask.  During  distillation,  however,  the  glass  stopper  is  replaced 
by  a  cork  with  a  double  perforation,  the  glass  tube  from  one  perforation  passing 
to  the  condenser,  while  the  bulbs  containing  water,  before  mentioned,  are  attached 
by  means  of  the  tube  in  the  other  perforation,  .\llow  the  distillation  process  to  pro- 
ceed until  practically  all  of  the  fluid  has  passed  over,  then  remove  the  receiving 
flask  and  insert  the  glass  stopper.     Xow  treat  the  distillate  carefully  with  10  c.c. 


536 


PHYSIOLOGICAL    CHEMISTRY 


of  a  N/io  solution  of  iodine  and  add  an  excess  of  sodium  hydroxide  solution  (15-25 
c.c.  of  20  per  cent).  Stopper  the  flask  and  shake  it  for  about  one  minute.  Add 
1-2  drops  of  concentrated  hydrochloric  acid,  and  note  the  production  of  a  brown 
color  at  the  point  of  contact  if  an  excess  of  iodine  is  present.  In  case  there  is  no 
such  excess,  the  solution  should  be  treated  with  N/io  iodine  solution  until  an  excess 
is  obtained.  Retitrate  this  excess  of  iodine  with  N/io  sodium  thiosulphate  solu- 
tion until  a  light  yellow  color  is  observed.  At  this  point  a  few  cubic  centimeters 
of  starch  paste  should  be  added  and  the  mixture  again  titrated  until  no  blue  color 
is  visible.     This  is  the  end-reaction. 

Calculation. — Subtract  the  number  of  cubic  centimeters  of  X/io  thiosulphate 
solution  used  from  the  volume  of  N/io  iodine  solution  employed.  Since  i  c.c.  of 
the  iodine  solution  is  equivalent  to  0.967  mg.  of  acetone,  and  since  i  c.c.  of  the  thio- 
sulphate solution  is  equivalent  to  i  c.c.  of  the  iodine  solution,  if  we  multiply  the 
remainder  from  the  above  subtraction  by  0.967  we  will  obtain  the  number  of  milli- 
grams of  acetone  in  the  100  c.c.  of  urine  examined. 

Calculate  the  quantity  of  acetone  in  the  twenty-four-hour  urine  specimen. 

Interpretation. — See  page  534. 

3.  Method  of  Scott-Wilson.^ — Principle. — The  urine  is  distilled 
with  acid  and  the  acetone  (preformed  and  from  acetoacetic  acid  by 


Fig.  •168. — ScoTT-WiLsoN  Apparatus. 


hydrolysis)  collected  in  an  alkaline  solution  of  basic  mercuric  cyanide. 
A  precipitate  of  keto-mercuric  cyanide  is  formed  according  to  the 
following  equation: 

C3H6O  +  2Hg(CN)2  +  3HgO  =  C30Hg5C4N4  +  3H2O 

The  precipitate  is  filtered  off  and  its  mercury  content  determined  by 
titration  with  potassium  thiocyanate  solution.  The  method  is  adapted 
to  the  determination  of  acetone  in  small  amounts,  and  has  the  ad- 
vantage over  the  iodoform  method  that  it  is  not  affected  by  alcohol. 

Procedure. — The  apparatus  for  the  distillation  of  acetone  is  set  up  as  in  the 
illustration  (Fig.  168).     Introduce  100  c.c.  of  the  normal  urine  (or  a  lesser  quan- 

'  Scott- Wilson:  Jour.  Physiol.,  42,  444,  191 1. 


URINE  537 

tity  of  an  abnormal  urine  high  in  acetone)  into  flask  A,  together  with  25  grams 
anhydrous  sodium  sulphate  and  i  c.c.  of  concentrated  sulphuric  acid.  Ten  c.c. 
of  40  per  cent  sodiimi  hydroxide  solution  are  placed  in  flask  C  and  10  c.c.  or  more 
of  the  mercuric  cyanide  solution'  in  the  Erlenmeyer  flask  E,  the  tube  D  being 
arranged  so  as  to  dip  beneath  the  surface  of  the  reagent. 

If  more  than  0.4  mg.  of  acetone  is  present  in  the  amount  of  urine  taken,  the 
quantity  of  reagent  should  be  proportionately  increased.  It  is  desirable  to  make 
a  test  to  determine  the  approximate  amount  of  acetone  bodies  as  outUned  under 
the  Folin-Hart  method  'page  534).  Connect  up  the  apparatus  closing  B  with  a 
glass-rod  plug,  and  hght  the  burners  under  A  and  C.  The  sodium  hydroxide 
solution  in  C  must  boil  before  the  urine  in  A  as  otherwise  condensation  would 
take  place  in  C.  Keep  the  hydroxide  solution  just  at  the  boiUng  point  and  aUow 
A  to  boil  briskly.  Note  the  first  appearance  of  turbidity  in  flask  E  and  distil  for 
five  minutes  from  that  time.  Remove  the  plug  at  B  and  extinguish  the  burners. 
Remove  tube  D  and  rinse  with  distilled  water  into  flask  E.  Allow  to  stand  for 
ten  minutes. 

Filter  off  the  precipitate  on  an  asbestos  mat  in  a  Gooch  crucible.  The  pores 
of  the  filter  should  have  been  partially  closed  by  filtering  a  suspension  of  talc 
through  it.  If  the  first  portions  of  the  filtrate  are  not  clear,  pass  them  through 
the  filter  again.  Transfer  any  precipitate  adherent  to  the  sides  of  the  flask  to  the 
filter  with  the  aid  of  water  and  wash  the  filter  thoroughly  with  water.  Transfer 
the  asbestos  and  precipitate  to  an  Erlenmeyer  flask  by  means  of  a  glass  rod. 
Any  precipitate  adherent  to  the  crucible  or  glass  rod  is  washed  into  the  flask  with 
a  jet  of  "acid  mixtiire"  (nitric  acid  40  parts,  sulphuric  acid  5  parts  and  water  55 
parts).  About  10  c.c.  of  this  acid  mixture  should  be  used  altogether.  Then  add 
I  c.c.  of  N  '5  potassium  permanganate  solution.-  Boil  until  the  brown  color  given 
by  the  permanganate  has  completely  disappeared.  This  should  occur  in  one  to 
two  minutes  but  if  the  decolorization  occurs  more  rapidly  (a  few  seconds), 
insufficient  permanganate  has  been  added  and  another  cubic  centimeter  or  so 
should  be  run  in  so  that  the  color  is  not  dispelled  on  two  minutes  boiling.  Clear 
with  a  few  drops  of  yellow  nitric  acid. 

Cool  the  completely  decolorized  solution  under  the  tap  and  add  2  c.c.  of  a 
saturated  solution  of  ferric  alum.  Titrate  with  standard  potassium  thiocyanate 
solution'  until  a  brownish  tinge  appears  throughout  the  solution.  Care  should  be 
taken  with  the  end  point  as  the  addition  of  several  drops  after  the  end  point  has 
been  reached  will  not  perceptibly  darken  the  color. 

Calculation. — One  c.c.  of  the  thiocyanate  solution  if  made  up  exactly  is 
equivalent  to  i  mg.  of  Hg  or  0.058  mg.  of  acetone.  Multiply  the  number 
of  cubic  centimeters  of  thiocyanate  used  by  its  equivalent  of  mercury  in  milli- 
grams and  by  0.058  to  get  the  amount  of  acetone  and  acetoacetic  acid  (expressed 
as  acetone)  in  the  amount  of  urine  analyzed.  Calculate  the  daily  output  from  the 
24-hour  volume. 

^  Scott-Wilson  Reagent. — Dissolve  0.5  gram  mercuric  cyanide  and  9  grams  sodium  hy- 
droxide in  60  c.c.  of  water  and  then  run  in  with  constant  stirring  20  c.c.  of  0.726S  per  cent 
silver  nitrate  solution.  (The  silver  nitrate  solution  is  made  by  taking  i  part  of  standard 
silver  nitrate  solution  (i  c.c.  =  10  mg.  XaCl)  and  3  parts  of  water.) 

2  Made  by  dissolving  6.324  grams  of  potassium  permanganate  in  water  and  making  up 
to  a  liter. 

'  Make  up  an  appro.ximately  o.i  per  cent  solution  of  potassium  thiocyanate  and  stand- 
ardize it  against  mercuric  nitrate  or  silver  nitrate.  It  is  convenient  to  have  the  solution 
of  such  a  strength  that  i  c.c.  =  i  mg.  of  Hg. 


538  PHYSIOLOGICAL   CHEMISTRY 

Interpretation. — See  page  534. 

Acetone 

Folin's  Method. — Principle. — The  preformed  acetone  is  aspirated 
from  the  urine  mixture  at  room  temperature  to  prevent  decomposition 
of  acetoacetic  acid.  The  acetone  is  collected  in  alkaline  hypoiodite 
solution  as  in  the  Folin-Hart  method  (page  533) .  Iodoform  is  formed 
quantitatively  and  the  excess  of  iodine  is  titrated  with  sodium  thio- 
sulphate. 

Procedure. — ^The  same  type  of  apparatus  is  used  in  this  method  as  that 
described  in  Folin's  method  for  the  determination  of  ammonia  (see  page  499). 
Introduce  20-25  c.c.  of  the  urine  under  examination  into  the  aerometer  cylinder 
and  add  10  drops  of  10  per  cent  phosphoric  acid/  8-10  grams  of  soditun  chloride,^ 
and  a  little  petroleum.  Introduce  into  an  absorption  flask,'  such  as  is  used  in  the 
ammonia  determination  (see  page  499),  150  c.c.  of  water,  10  c.c.  of  a  40  per  cent 
solution  of  potassixim  hydroxide,  and  an  excess  of  a  N/io  iodine  solution.  Con- 
nect the  flask  with  the  aerometer  cyUnder,  attach  a  Chapman  pump,  and  permit 
an  air  current,  slightly  less  rapid  than  that  used  for  the  determination  of  ammonia, 
to  be  drawn  through  the  solution  for  20-25  minutes.  All  of  the  acetone  will,  at 
this  point,  have  been  converted  into  iodoform  in  the  absorption  flask.  Add 
10  c.c.  of  concentrated  hydrochloric  acid  (a  volimie  equivalent  to  that  of  the  strong 
alkali  originally  added),  to  the  contents  of  the  latter  and  titrate  the  excess  of 
iodine  by  means  of  N/io  sodimn  thiosulphate  solution  and  starch,  as  in  the  Mes- 
singer-Huppert  method  (see  page  535). 

Folin  has  further  made  suggestions  regarding  the  simultaneous  determination 
of  acetone  and  ammonia  by  the  use  of  the  same  air  current.*  This  is  an  important 
consideration  for  the  clinician  inasmuch  as  urines  which  contain  acetone  and  aceto- 
acetic acid  are  generally  those  from  which  the  ammonia  data  are  also  desired.  The 
procedure  for  the  combination  method  is  as  follows:  Arrange  the  ammonia  appar- 
atus as  usual  (see  page  499),  and  to  the  aerometer  of  the  ammonia  apparatus  attach 
the  acetone  apparatus  set  up  as  described  above.  Regulate  the  air  current  with 
special  reference  to  the  determination  of  acetone  and  at  the  end  of  20-25  minutes 
disconnect  the  acetone  apparatus  and  complete  the  determination  of  the  acetone 
as  just  described.  The  air  current  is  not  interrupted,  and  after  having  run  one 
and  one-half  hours  the  ammonia  apparatus  is  detached  and  the  ammonia  determina- 
tion completed  as  described  on  page  500. 

If  data  regarding  acetoacetic  acid  are  desired,  the  result  obtained  by  Folin's 
method  may  be  subtracted  from  the  result  obtained  by  the  Messinger-Huppert 
method  (see  page  535),  inasmuch  as  the  latter  method  determines  both  acetone  and 
acetoacetic  acid.  Under  all  conditions  the  determination  of  acetone  should  be  as 
expeditious  as  possible.     This  is  essential,  not  only  because  of  the  fact  that  any 

1  Oxalic  acid  (0.2—0.3  gram)  may  be  substituted  if  desired. 

2  Acetone  is  insoluble  in  a  saturated  solution  of  sodium  chloride. 

'  Folin's  improved  absorption  tube  (see  Fig.  128,  p.  500)  should  be  used  in  this  connec- 
tion inasmuch  as  the  original  type  embracing  the  use  of  a  rubber  stopper  is  unsatisfactory 
because  of  the  solvent  action  of  alkaline  hypoiodite  on  rubber. 

*  These  determinations  may  even  be  made  on  the  same  sample  of  urine  if  the  sample  is 
too  small  for  the  double  determination. 


URINE  539 

acetoacetic  acid  present  in  the  urine  will  become  transformed  into  acetone,  but  also 
because  of  the  rapid  spontaneous  decomposition  of  the  alkaline  hypoiodite  solution 
used  in  the  determination  of  the  acetone.  It  has  been  claimed  that  alkaline  hypo- 
iodite solutions  are  almost  completely  converted  into  iodale  solutions  in  one-half 
hour.  Folin  states,  however,  that  the  transformation  is  not  so  rapid  as  this,  but 
he  nevertheless  emphasizes  the  necessity  of  rapidity  of  manipulation.  At  the  same 
time  it  should  be  remembered  that  the  air  current  must  not  be  as  rapid  as  for  am- 
monia, inasmuch  as  the  alkaline  hypoiodite  solution  will  not  absorb  all  the  acetone 
under  those  conditions. 

Interpretation.— \Js,u.a.\\y  about  one-fourth  of  the  total  acetone  and 
acetoacetic  acid  excretion  is  in  the-  form  of  acetone,  but  the  proportion 
varies  considerably-     See  Acetone  and  Acetoacetic  Acid. 

Acetoacetic  Acid 

1.  Folin-Hart  Method. — Arrange  the  apparatus  as  described  under  the 
Folin-Hart  method  for  the  determination  of  acetone  and  acetoacetic  acid  (see 
page  533).  Start  the  air  current  in  the  usual  way  and  permit  it  to  nin  25  minutes 
without  the  application  of  heat  to  the  urine  under  examination.  Under  these 
conditions  the  preformed  acetone  present  in  the  solution  is  all  removed  'see 
page  538).  Immediately  attach  a  freshly  prepared  absorption  bottle  or  intro- 
duce fresh  alkaline  hypoiodite  solution  into  the  original  bottle.  Apply  heat  to 
the  large  test-tube  as  akeady  described  (see  page  533),  in  order  to  convert  the 
acetoacetic  acid  into  acetone,  permit  the  air  current  to  continue  for  the  usual 
25-minute  period,  and  determine  the  acetoacetic  acid  value  in  terms  of  acetone 
by  the  usual  titration  procedure  (see  page  5361. 

Interpretation. — Ordinarily  about  three-fourths  of  the  total  acetone 
and  acetoacetic  acid  excretion  occurs  in  the  form  of  acetoacetic  acid, 
which,  however,  is  readily  transformed  into  acetone  with  loss  of  carbon 
dioxide.     See  Acetone  and  Acetoacetic  Acid. 

2.  Folin-Messinger-Huppert  and  Folin-Scott-Wilson  Method. — Determine 
the  combined  acetone  and  acetoacetic  acid,  in  terms  of  acetone,  by  the  Messinger- 
Huppert  method  or  the  Scott -Wilson  method  (see  pages  535  and  536  ^  and  sub- 
sequently determine  the  acetone  by  FoUn's  method  (see  page  5381.  Subtract 
the  value  determined  by  the  second  method  from  that  obtained  in  either  of  the 
first  two  methods  to  secure  data  regarding  the  acetoacetic  acid  content  of  the 
urine,  in  terms  of  acetone. 

Acetone,  Acetoacetic  Acid,  and  ^-Hydroxybutyric  Acid 

I.  Method  of  Shaffer  and  Mairiott.^—P rincifylc.  By  this  procedure 
the  combined  acetone  and  acetoacetic  acid  is  determined  in  the  same 
sample  of  urine  used  in  the  determination  of  /3-hydroxybutyric  acid. 
The  preformed  acetone  and  the  acetoacetic  acid  are  distilled  off  to- 

'  Shaffer  and  Marriott:  Jour.  Biol.  Chein.,  16,  265,  1915. 


54©  PHYSIOLOGICAL   CHEMISTRY 

gether  as  acetone  and  determined  by  the  iodine  titration  method. 
The  )8-hydroxybutyric  acid  remains  in  the  residue  from  distillation 
and  is  oxidized  by  means  of  potassium  bichromate.  The  product 
of  the  oxidation  is  acetone  which  is  distilled  off  and  determined  as 
such. 

Procedure. — Determination  of  Acetone  and  Acetoacetic  Acid. — Measure 
from  25-100  c.c.^  or  more  of  urine  (usually  50  c.c.)  with  a  pipette  into  a  500  c.c. 
volumetric  flask  containing  200-300  c.c.  of  water.  Add  basic  lead  acetate 
solution  (U.  S.  P.)  in  volume  equal  to  that  of  the  urine  used-  and  mix  well.  Add 
concentrated  ammonium  hydroxide  in  amount  equal  to  about  one-half  that  of 
the  lead  acetate  solution.  Dilute  the  contents  of  the  flask  to  the  mark  with 
water,  shake,  and  after  standing  a  few  minutes  filter  the  liquid,  preferably 
through  a  folded  filter.  Measure  200  c.c.  of  the  filtrate  into  a  round  bottom 
flask  (800  c.c.  or  liter  Kjeldahl  flasks  are  convenient)  dilute  with  water  to  about 
600  c.c.  and  add  15  c.c.  of  concentrated  sulphuric  acid  and  a  little  talc  or  a  boiling 
stone.  Distil  until  about  200  c.c.  of  distillate  have  been  collected.  The  tube 
of  the  condenser  should  dip  beneath  the  surface  of  the  water  in  the  receiving 
flask  so  that  no  loss  of  acetone  will  occur.  The  distiUing  flask  must  also  be 
fitted  with  a  dropping  tube  or  dropping  funnel  so  water  may  be  run  in  from  time 
to  time  and  the  volinne  of  liquid  in  the  flask  kept  from  becoming  less  than 
400-500  c.c.  A  good  condenser  should  be  used,  but  it  is  not  necessary  to  cool 
the  distillate  in  ice. 

The  distillate  thus  obtained  is  transferred  to  a  second  Kjeldahl  flask  and 
10  c.c.  of  10  per  cent  NaOH  added.  It  is  then  redistilled  for  about  20  minutes.^ 
This  second  distillate  is  then  titrated  with  standard  iodine  and  thiosulphate 
solution  as  in  the  method  for  acetone  and  acetoacetic  acid  previously  given 
(see  Messinger-Huppert  method,  page  535),  and  the  calculation  made  in  the 
same  way.  The  result  gives  the  combined  acetone  and  acetoacetic  acid  content 
of  the  urine  expressed  as  acetone. 

Determination  of  the  /3-Hydroxybutyric  Acid. — The  flask  containing  the 
residue  from  the  first  distillation  above  is  used  in  the  determination  of  the 
^-hydroxybutyric  acid.  A  new  receiver  is  arranged  as  before  with  the  tip  from  the 
condenser  dipping  beneath  the  surface  of  the  water.  The  distillation  is  then 
continued  and  water  added  whenever  necessary  to  keep  the  volume  between 
400  and  600  c.c.  A  dilute  solution  (i  per  cent)  of  potassiimi  bichromate  is  added 
during  the  distillation.  At  first  20  c.c.  of  this  i  per  cent  solution  is  added  slowly 
through  the  dropping  tube  and  then  10  c.c.  portions  every  15-20  minutes  until 

1  The  amount  used  depends  upon  the  expected  yield  of  /3-oxybutyric  acid.  In  the  case 
of  urines  which  give  a  strong  ferric  chloride  reaction  for  diacetic  acid,  or  when  5-10  grams 
or  more  of  /3-oxybutyric  acid  is  expected,  it  is  unnecessary  to  use  more  than  25-50  c.c.  of 
urine.  However,  in  case  only  a  trace  of  /3-oxybutyric  acid  is  expected,  the  volume  should  be 
much  larger  as  indicated.  Under  all  conditions,  the  amount  specified  is  sufficient  for  dupli- 
cate determinations.  It  is  desirable  to  use  such  a  volume  of  urine  as  contains  the  proper 
amount  of  /3-oxybutyric  acid  to  yield  25-50  mg.  of  acetone. 

2  If  the  urine  contains  but  little  or  no  sugar  only  half  the  amount  or  less  of  lead  acetate 
should  be  used. 

2  In  many  instances  when  a  high  degree  of  accuracy  is  not  required  this  redistillation 
may  be  omitted  and  the  first  distillate  titrated  directly.  The  results  so  obtained  are 
slightly  higher  than  those  after  redistillation  from  alkali.  The  object  of  the  redistillation 
is  to  get  rid  of  fatty  acids  of  which  formic  acid  is  one  of  the  most  troublesome. 


URINE  541 

the  whole  has  been  added.  ^  Should  the  liquid  become  markedly  green  the 
bichromate  must  be  added  at  correspondingly  shorter  intervals  and  in  amount 
suflBcient  to  maintain  a  sUght  red-yellow  color  of  the  chromic  acid  which  may  be 
detected  even  in  the  presence  of  the  green.  Continue  the  distillation  with  moder- 
ate boihng  for  from  two  to  three  hours.  The  distillate  which  should  be  collected 
in  a  liter  flask  to  avoid  transference  is  again  distilled  for  about  20  minutes 
after  adding  10  c.c.  of  10  per  cent  sodium  hydroxide  and  25  c.c.  of  3  per  cent 
hydrogen  peroxide.  The  flask  must  be  heated  cautiously  until  the  peroxide  has 
been  decomposed.  This  final  distillate  is  titrated  with  standard  iodine  and 
thiosulphate  in  the  usual  manner  (see  page  536;  and  the  result  expressed  as 
hydroxybutyric  acid.  One  c.c.  of  N/ 10  iodine  solution  is  equivalent  to  1.736 
mg.  of  hydroxybutyric  acid.  One  c.c.  of  the  1.035  N/io  iodine  which  is  recom- 
mended for  acetone  titrations  is  equivalent  to  1.793  n^g-  of  hydroxybutyric  acid. 
About  10  per  cent  should  be  added  to  the  results  for  /3-hydroxybutyric  acid  as 
obtained  by  this  method  as  the  yield  of  acetone  is  only  about  90  per  cent  of 
the  theoretical.  This  error  appears  to  be  practically  constant,  so  that  satisfactory 
results  may  be  obtained  by  correction. 

Interpretation. — i3-Hydroxybutyric  acid  may  occur  in  normal  human 
urine  to  the  extent  of  20-30  mg.  per  day.  In  fasting  or  on  a  carbo- 
hydrate-free diet  very  large  amounts  may  be  excreted  (up  to  20  grams 
per  day).  In  severe  diabetes  mellitus  the  largest  amounts  are  found. 
Excretions  of  50  or  even  100  grams  or  over  per  day  have  been  noted. 
It  is  always  'present  in  the  urine  when  large  amounts  of  acetone  are 
present.  In  severe  diabetes  it  is  usually  the  most  abundant  of  the 
acetone  bodies  making  up  from  60-80  per  cent  of  the  total.  The 
ratio  is,  however,  by  no  means  constant  and  it  should  be  borne  in  mind 
that  in  rare  cases  large  amounts  of  /3-hydroxybutyric  acid  may  be 
eliminated  although  the  acetone  excretion  is  very  low.  (See  Acetone 
and  Acetoacetic  Acid.) 

2.  Nephelometric  Methods  for  Acetone,  Acetoacetic  Acid,  and 
/3-Hydroxybutyric  Acid. — Folin  and  Denis'-  have  suggested  nephelo- 
metric methods  for  the  determination  of  the  acetone  bodies  in  urine. 
The  methods  are  similar  to  those  used  by  Marriott  for  blood  analysis 
(see  Chapter  XVI)  but  the  air  current  is  used  instead  of  distillation. 

/3 -Hydroxybutyric  Acid 

I.  Black's  Method  —  Principle. — The  urine  is  mixed  with  plaster 
of  Paris  to  form  a  coarse  meal  and  extracted  with  ether  to  obtain 
the  hydr()xybut\ric  acid  which  is  then  determined  by  means  of  the 
polariscope. 

^  From  0.5  gram  to  i  gram  of  bichromate  will  usually  be  sufficient,  and  not  more  than 
I  gram  should  be  added  unless  the  liquid  turns  Rreen  indicatinn  a  preat  reduction  to 
chromium  sulphate.  Very  rarely  2  or  3  grams  of  bichromate  may  be  necessary,  especially 
if  the  sugar  has  not  been  completely  removed. 

^  Folin  and  Denis:  Jour.  Biol.  Cliem.,  18,  263,  1914. 


542  PHYSIOLOGICAL    CHEMISTRY 

Procedure. — Render  50  c.c.  of  the  urine  under  examination  faintly  alkaline 
with  sodium  carbonate  and  evaporate  to  one-third  the  original  volume.  Con- 
centrate to  about  10  c.c.  on  a  water-bath,  cool  the  residue,  acidify  it  with  a  few 
drops  of  concentrated  hydrochloric  acid^  and  add  plaster  of  Paris  to  form  a 
thick  paste.  Permit  the  mixture  to  stand  until  it  begins  to  "set,"  then  break 
it  up  with  a  stout  glass  rod  having  a  blunt  end  and  reduce  the  material  to  the 
consistency  of  a  fairly  dry  coarse  meal.-  Transfer  the  meal  to  a  Soxhlet  appa- 
ratus and  extract  with  ether  for  six  to  ten  hours.  At  the  end  of  this  period 
evaporate  the  ether-extract  either  spontaneously  or  in  an  air  current.  Dissolve 
the  residue  in  water,  add  a  httle  boneblack,  if  necessary,  filter  until  a  clear 
solution  is  obtained  and  make  up  the  filtrate  to  a  known  volvmie  (25  c.c.  or  less) 
with  water.  The  /3-hydroxybutyric  acid  should  then  be  determined  by  means  of 
the  polariscope.     Its  specific  rotation  is  —24.12. 

This  method  is  satisfactory  where  the  amount  of  hydroxybutyric 
acid  present  is  not  too  small.  Errors  due  to  incomplete  extraction  of 
the  acid  are  partly  counterbalanced  by  the  extraction  of  other  levo- 
rotatory  substances. 

Interpretation. — See  page  541. 

2.  Method  of  Schaffer  and  Marriott. — See  page  539. 

Indican 

Ellinger's  Method. — Principle. — This  method  for  the  quantitative 
determination  of  indican  is  based  upon  the  principle  underlying  Jaffe's 
qualitative  test  for  indican.  The  urine  after  removal  of  interfering 
substances  with  basic  lead  acetate  is  treated  with  Obermayer's  reagent 
to  oxidize  the  indican  to  indigo.  The  indigo  is  extracted  with  chloro- 
form, the  chloroform  evaporated  off  and  the  residue  titrated  with 
potassium  permanganate.  The  method  is  not  very  accurate  but  is  as 
satisfactory  as  any. 

Procedure. — To  50  c.c.  of  urine^  in  a  small  beaker  or  casserole  add  5  c.c. 
of  basic  lead  acetate  solution,*  mix  thoroughly,  and  filter.  Transfer  40  c.c.  of 
the  filtrate  to  a  separatory  funnel,  add  an  equal  volmne  of  Obermayer's  reagent 
(see  page  388)  and  20  c.c.  of  chloroform,  and  extract  in  the  usual  manner.  This 
extraction  with  chloroform  should  be  repeated  untU  the  chloroform  solution 
remains  colorless.  Shake  up  the  combined  chloroform  extracts  two  or  three 
times  with  distilled  water  in  a  separating  funnel  and  complete  the  purification 
by  extracting  with  very  dilute  sodium  hydroxide  (i:  1000).  Remove  all  traces 
of  alkaU  by  washing  with  water.  Now  filter  the  combined  chloroform  extracts 
through  a  dry  filter  paper  into  a  dry  Erlenmeyer  flask.  Distil  off  the  chloro- 
form, heat  the  residue  on  a  boihng  water-bath  for  five  minutes  in  the  open 

^  The  residue  should  give  a  distinct  red  color  with  litmus  paper. 

^  Before  this  is  accomplished  it  may,  in  some  cases,  be  necessary  to  add  a  little  more 
plaster  of  Paris. 

'  If  the  urine  under  examination  is  neutral  or  alkaline  in  reaction  it  may  be  made 
faintly  acid  with  acetic  acid  before  adding  the  basic  lead  acetate. 

*  For  preparation  of  basic  lead  acetate  solution  see  Appendix. 


URINE  543 

flask,  and  wash  the  dried  residue  with  hot  water.'  Add  lo  c.c.  of  concentrated 
sulphuric  acid  to  the  washed  residue,  heat  on  the  water-bath  for  five  to  ten 
minutes,  dilute  with  loo  c.c.  of  water,  and  titrate  the  blue  solution  with  a  very 
dilute  solution  of  potassium  permanganate.-  The  end  point  is  indicated  by  the 
dissipation  of  all  the  blue  color  from  the  solution  and  the  formation  of  a  pale 
yellow  color. 

Beautiful  plates  of  indigo  blue  sometimes  appear  in  the  chloroform  extract 
of  urines  containing  abundant  indican.  In  urines  preserved  by  thymol  the 
determination  of  indican  is  interfered  with  unless  great  care  is  taken  in  washing 
the  chloroform  extract  with  dilute  alkah.  Care  should  be  taken,  therefore,  to 
make  the  indican  determination  upon  fresh  urine,  before  the  addition  of  the 
preservative. 

Plasencia^  has  suggested  a  method  which  is  shorter  than  Ellinger's  and  ac- 
cording to  its  sponsor,  just  as  accurate. 

Calculation. — One  cubic  centimeter  of  the  diluted  permanganate  solution 
is  equivalent  to  about  0.15  mg.  of  indigo.  Ellinger  claims  that  one-sixth  of  the 
amount  determined  must  be  added  to  the  value  obtained  by  titration  in  order  to 
secure  accurate  data.    This  correction  should  always  be  made. 

Interpretation. — From  4-20  mg.  of  indican  are  excreted  per  day  by 
normal  men.  In  normal  individuals  the  variations  are  dependent 
mainly  upon  the  diet.  A  meat  diet  increases  the  indican  excretion, 
while  a  milk  or  carbohydrate-rich  diet  decreases  it.  Pathologically 
the  greatest  increases  are  found  in  disorders  involving  increased 
putrefaction  and  stagnation  of  intestinal  contents.  Bacterial  de- 
composition of  body  protein  as  in  gangrene,  putrid  pus  formation,  etc., 
gives  rise  to  increases. 

Phenols 

Colorimetric  Method  of  Folin  and  Denis. ^ — Principle.- — This  method 
is  based  upon  the  fact  that  phenols  yield  with  a  solution  of  phospho- 
tungstic-phosphomolybdic  acid  and  alkali  a  deep  blue  color  the  depth 
of  which  is  proportional  to  the  amount  of  such  substances  present. 
Traces  of  protein,  which  may  be  present  in  the  urine,  and  uric  add 
give  a  blue  color  with  the  reagent  and  are  removed  by  precipitation 
with  an  ammoniacal  silver  solution  and  colloidal  iron  as  a  preUminary 
to  the  determination  of  the  phenols. 

Procedure. — Removal  of  Interfering  Substances.-  Place  10  c.c.  of  ordinary 
urine,  or  20  c.c.  of  a  dilute  urine  in  a  50  c.c.  volumetric  flask.     To  this  add  an 

*  The  washing  should  be  continued  until  the  wash  water  is  no  longer  colored.  Ordi- 
narily two  or  three  washings  are  sullicient.  If  a  separation  of  indigo  particles  takes  place 
during  the  washing  process,  the  wash  water  should  be  filtered,  the  indigo  extracted  with 
chloroform,  and  the  usual  method  applied  from  this  point. 

^  A  "stock  solution"  of  potassium  permanganate  containing  3  grams  per  liter  should  be 
prepared,  and  when  needed  for  titration  purposes  a  suitable  volume  of  this  solution  should 
be  diluted  with  40  volumes  of  water.  The  potassium  permanganate  soliition  may  be 
standardized  with  pure  indigo. 

'  Plasencia:  Revista  de  Medicina  y  Cirugia.,  17,  r,  1912. 

*  Folin  and  Denis:  J.  Biol.  Ghent.,  22,  305,  191 5. 


544  PHYSIOLOGICAL   CHEMISTRY 

acid  silver  lactate  solution  (from  2  to  20  c.c.  of  a  3  per  cent  solution  of  silver 
lactate  in  3  per  cent  lactic  acid)  until  no  further  precipitate  is  obtained.  Add 
a  few  drops  of  colloidal  iron,  shake  the  flask,  dilute  to  mark  with  distilled  water, 
shake  again,  and  filter  the  contents  through  a  dry  filter.  Phenols  are  not  pre- 
cipitated by  this  procedure  but  are  recovered  quantitatively  in  the  filtrate.  Trans- 
fer 25  c.c.  of  the  filtrate  to  a  50  c.c.  volumetric  flask,  and  add  a  sufficient  quantity 
of  saturated  sodium  chloride  solution,  containing  10  c.c.  of  strong  hydrochloric 
acid  per  liter,  to  precipitate  all  the  silver.  Fill  the  flask  to  the  mark  with  dis- 
tilled water,  mix  thoroughly,  and  filter  through  a  dry  filter.  This  filtrate,  which 
contains  half  the  phenol  from  the  urine  taken  for  analysis,  is  used  for  the  deter- 
mination of  free  and  total  phenols. 

Free  Phenols. — Place  20  c.c.  of  the  filtrate  mentioned  above  in  a  50  c.c. 
volumetric  flask,  add  5  c.c.  of  the  phosphotungstic-phosphomolybdic  acid  re- 
agent^ and  15  c.c.  of  a  saturated  solution  of  sodium  carbonate.  Dilute  to  volume 
with  luke  warm  water  (30-35°C.),  mix  thoroughly  and  after  allowing  to  stand 
for  20  minutes  compare  the  deep  blue  color  in  the  Duboscq  colorimeter  (see 
Fig'  i53>  page  486)  against  a  standard  solution  of  phenol  (see  below)  similarly 
treated. 

Total  Phenols  (Free  and  Conjugated). — Place  20  c.c.  of  the  same  filtrate 
used  for  the  determination  of  free  phenols  in  a  large  test-tube,  add  10  drops 
of  concentrated  hydrochloric  acid,  cover  the  tube  with  a  small  funnel,  heat 
rapidly  to  boiUng  over  a  free  flame,  and  then  place  in  a  boiling  water-bath 
for  ten  minutes.  This  process  serves  to  decompose  the  conjugated  phenols. 
At  the  end  of  the  ten  minutes,  remove  the  tube,  cool,  and  transfer  the  contents 
to  a  100  c.c.  volumetric  flask.  Add  lo  c.c.  of  the  phosphotungstic-phospho- 
molybdic  reagent,  25  c.c.  of  saturated  sodium  carbonate  solution,  dilute  to  mark 
with  luke  warm  water  (30-35°C.),  mix  thoroughly,  allow  to  stand  for  20  minutes, 
and  read  in  the  Duboscq  colorimeter  (see  page  486)  against  a  standard  solution 
of  phenol  (see  below). 

Standard  Solution  of  Phenol. — The  standard  used  is  a  solution  of  pure  phenol 
in  N/ioo  hydrochloric  acid  containing  i  mg.  of  phenol  in  10  c.c,  standardized  by 
means  of  the  iodometric  titration.  The  preparation  is  carried  out  as  follows:  Make 
a  phenol  solution  in  N/io  hydrochloric  acid,  which  contains  approximately  i  mg.  of 
crystallized  phenol  per  cubic  centimeter.  Transfer  25  c.c.  of  this  solution  to  a 
250  c.c.  flask,  add  50  c.c.  of  N/io  sodium  hydroxide,  heat  to  65°C.,  add  25  c.c.  of 
N/io  iodine  solution,  stopper  the  flask,  and  let  stand  at  room  temperature  30  or 
40  minutes.  Add  5  c.c.  of  concentrated  hydrochloric  acid  and  titrate  the  excess 
of  iodine  with  N/io  thiosulphate  solution.  Each  cubic  centimeter  of  N/io  iodine 
solution  corresponds  to  1.567  mg.  of  phenol.  On  the  basis  of  the  result  dilute  the 
phenol  solution  so  that  10  c.c.  contain  i  mg.  of  phenol.  Five  c.c.  of  this  solution 
(equivalent  to  0.5  mg.  of  phenol),  when  10  c.c.  of  the  phosphotungstic  phospho- 
molybdic  reagent  and  25  c.c.  of  saturated  sodium  carbonate  solution  are  added,  and 
the  whole  made  up  with  water  at  about  3o°C.  to  100  c.c,  give  when  set  in  the 
colorimeter  at  20  mm.  a  convenient  standard. 

Calculation. — The  filtrate  used  for  the  determination  of  free  and  total  phenols 

^  This  reagent  is  prepared  as  follows:  Boil  together  for  two  hours  (using  a  reflux  con- 
denser) 100  grams  of  sodium  tungstate,  20  grams  of  phosphomolybdic  acid  (or  an  equiva- 
lent of  molybdic  acid),  50  c.c.  of  phosphoric  acid  (85  per  cent.),  and  75  c.c.  of  distilled 
water.  After  the  period  of  heating,  cool,  dilute  to  i  liter  with  distilled  water,  and  filter 
If  necessary. 


URINE  545 

contains  the  phenols  from  one-half  the  amount  of  urine  analyzed.  The  actual 
determination  of  phenols,  both  free  and  total,  is  made  upon  a  two-fifths  portion 
of  this  filtrate  and  this  amount  of  filtrate  contains  the  phenols  from  one-fifth 
of  the  amount  of  urine  analyzed.  In  the  determination  of  free  phenols  the  colored 
solution  is  diluted  to  only  half  that  of  the  standard  while  in  the  determination 
of  total  phenols  the  dilution  is  the  same  as  that  of  the  standard. 
Hence, 

Ri 


and 


=  miUigrams  of  free  phenol 
R2  X  4 


=  miUigrams  of  total  phenol 
R2  X  2 


in  2  c.c.  or  4  c.c.  of  urine  according  to  whether  10  c.c.  or  20  c.c.  of  urine  was 
taken  for  analysis,  when  Ri  is  taken  as  the  reading  obtained  with  the  standard 
solution,  and  R2  is  taken  as  the  reading  obtained  with  the  unknown. 

Interpretation. — By  this  method  total  phenol  excretions  of  from 
0.2-0.5  gram  per  day  have  been  noted  in  normal  individuals.  These 
results  are  much  higher  than  figures  previously  obtained  by  other 
methods.  The  free  phenols  varied  from  0.1-0.3  gram  per  day.  The 
total  phenol  excretion  appears  to  vary  directly  but  not  proportionately 
with  the  protein  intake.  The  amount  of  conjugated  phenol  indicates 
the  extent  to  which  the  phenols  have  been  detoxicated.  The  excretion 
of  phenols  is  increased  in  gastro-intestinal  disorders  associated  with 
increased  putrefaction.  It  is  increased  by  the  ingestion  of  phenols  or 
of  benzene. 

Oxalic  Acid 

Salkowski-Autenrieth  and  Barth  Method. — Principle. — The  o.xalic  acid  is  pre- 
cipitated by  means  of  CaC^.  From  the  solution  of  this  precipitate  in  hydrochloric 
acid  the  oxalic  acid  is  extracted  with  ether  and  reprecipitated  as  calcium  oxalate. 

Procedure. — Place  the  24-hour  urine  specimen  in  a  precipitating  jar,  add  an 
excess  of  calcium  chloride,  render  the  urine  strongly  ammoniacal,  stir  it  well,  and 
allow  it  to  stand  18-20  hours.  Filter  oflf  the  precipitate,  wash  it  with  a  small 
amount  of  water  and  dissolve  it  in  about  30  c.c.  of  a  hot  15  per  cent  solution  of 
hydrochloric  acid.  By  means  of  a  separatory  funnel  extract  the  solution  with  150 
c.c.  of  ether  which  contains  3  per  cent  of  alcohol,  repeating  the  extraction  four  or 
five  times  with  fresh  portions  of  ether.  Unite  the  ethereal  extracts,  allow  them  to 
stand  for  an  hour  in  a  flask,  and  then  filter  through  a  dry  filter  paper.  Add  5  c.c. 
of  water  to  the  filtrate,  to  prevent  the  formation  of  diethyl  oxalate  when  the  solu- 
tion is  heated,  and  distil  ofif  the  ether.  If  necessary,  decolorize  the  Hquid  with 
animal  charcoal  and  filter.  Concentrate  the  filtrate  to  3-5  c.c,  add  a  Httle  calcium 
chloride  solution,  make  it  ammoniacal,  and  after  a  few  minutes  render  it  slightly 
acid  with  acetic  acid.  Allow  the  acidified  solution  to  stand  several  hours,  collect 
35 


546  PHYSIOLOGICAL   CHEMISTRY 

the  precipitate  of  calcium  oxalate  on  a  washed  filter  paper, ^  wash,  incinerate  strongly 
(to  CaO),  and  weigh  in  the  usual  manner. 

Calculation. — Since  56  parts  of  CaO 'are  equivalent  to  90  parts  of  oxalic  acid, 
the  quantity  of  oxalic  acid  in  the  volume  of  urine  taken  may  be  determined  by 
multiplying  the  weight  of  CaO  by  the  factor  1.6071. 

Interpretation. — From  15-20  mg.  of  oxalic  acid  are  excreted  by  a  normal  adult 
on  an  ordinary  mixed  diet.  It  arises  from  oxalates  of  the  food  ingested  and  from 
fat  and  protein  metabolism.  It  is  increased  by  the  ingestion  of  apples,  grapes, 
cabbage,  etc.,  although  most  of  the  ingested  oxalate  is  destroyed.  It  is  increased 
in  disturbances  of  metabolism  associated  with  decreased  oxidation,  according  to 
certain  observers.     The  term  "oxaluria"  has  been  largely  a  misnomer. 

Sulphur 
(a)  Gravimetric  Procedures 

I.  Total  Sulphates.^ — Folin's  Method. — Principle. — The  sulphuric 
acid  of  the  conjugated  sulphates  is  set  free  by  boiling  with  acid.  The 
total  sulphates  are  then  precipitated  with  barium  chloride. 

Procedure. — Place  25  c.c.  of  urine  in  a  200-250  c.c.  Erlemneyer  flask,  add 
20  c.c.  of  dilute  hydrochloric  acid^  (i  volume  of  concentrated  HCl  to  4  voltunes  of 
water)  and  gently  boil  the  mixture  for  20-30  minutes.  To  minimize  the  loss  of 
water  by  evaporation  the  mouth  of  the  flask  should  be  covered  with  a  small  watch 
glass  during  the  boiling  process.  Cool  the  flask  for  2-3  minutes  in  running  water, 
and  dilute  the  contents  to  about  150  c.c.  by  means  of  cold  water.  Add  10  c.c. 
of  a  5  per  cent  solution  of  baritmi  chloride  slowly,  drop  by  drop,  to  the  cold  solu- 
tion.^ The  contents  of  the  flask  should  not  be  stirred  or  shaken  during  the  addi- 
tion of  the  barium  chloride.  AUow  the  mixture  to  stand  at  least  one  hour,  then 
shake  up  the  solution  and  filter  it  through  a  weighed  Gooch  crucible.* 

Wash  the  precipitate  of  BaS04  with  about  250  c.c.  of  cold  water,  dry  it  in  an 
air-bath  or  over  a  very  low  flame,  then  ignite,*  cool  and  weigh. 

Calculation. — Subtract  the  weight  of  the  Gooch  crucible  from  the  weight  of 
the  crucible  and  the  BaSO^  percipitate  to  obtain  the  weight  of  the  precipitate. 

'  Schleicher  and  Schiill,  No.  5S9,  is  satisfactory. 

-  If  it  is  desired,  50  c.c.  of  urine  and  4  c.c.  of  concentrated  acid  may  be  used  instead. 

'  A  dropper  or  capillary  funnel  made  from  an  ordinary  calcium  chloride  tube  and  so 
constructed  as  to  deliver  10  c.c.  in  2-3  minutes  is  recommended  for  use  in  adding  the  barium 
chloride. 

*  If  a  Gooch  crucible  is  not  available,  the  precipitate  of  BaS04  may  be  filtered  off  upon 
a  washed  filter  paper  (Schleicher  &  SchuU's,  No.  589,  blue  ribbon),  and  after  washing  the 
precipitate  with  about  250  c.c.  of  cold  water  the  paper  and  precipitate  may  be  dried  in  an 
air-bath  or  over  a  low  flame.  The  ignition  may  then  be  carried  out  in  the  usual  way  in  the 
ordinary  platinum  or  porcelain  crucible.  In  this  case  correction  must  be  made  for  the 
weight  of  the  ash  of  the  filter  paper  used. 

5  Care  must  be  taken  in  the  ignition  of  precipitates  in  Gooch  crucibles.  The  flame 
should  never  be  applied  directly  to  the  perforated  bottom  or  to  the  sides  of  the  crucible,  since 
such  manipulation  is  invariably  attended  by  mechanical  losses.  The  crucibles  should 
always  be  provided  with  lids  and  tight  bottoms  during  the  ignition.  In  case  porcelain  Gooch 
crucibles,  whose  bottoms  are  not  provided  with  a  non-perforated  cap,  are  used,  the  crucible 
may  be  placed  upon  the  lid  of  an  ordinary  platinum  crucible  during  ignition.  The  lid 
should  be  supported  on  a  triangle,  the  crucible  placed  upon  the  lid  and  the  flame  applied 
to  the  improvised  bottom.  Ignition  should  be  complete  in  10  minutes  if  no  organic  matter 
is  present. 


URINE  547 

The  weight  of  SO^^  in  the  volume  of  urine  taken  may  be  determined  by  means  of 
the  following  proportion. 

Mol.  Wt.        Wt.  of  Mol.  wt. 

BaS04 :  BaSO^ :  :  SO3 :  x(wt.  of  SO3  in  grams). 

Representing  the  weight  of  the  BaS04  precipitate  by  y  and  substituting  the  proper 
molecular  weights,  we  have  the  following  proportion : 

233-43  :  y :  :  80.06  :  x  (wt.  of  SO3  in  grams  in  the  quantity  of  urine  used). 

Calculate  the  quantity  of  SO 3  in  the  twenty-four-hour  specimen  of  urine. 
To  express  the  result  in  percentage  of  SO3  simply  divide  the  value  of  x,  as  just 
determined,  by  the  quantity  of  urine  used. 

Interpretation. — The  total  sulphate  excretion  (ethereal  and  inorganic 
sulphates)  by  a  normal  adult  on  a  mixed  diet  is  usually  between  1.5  and 
3.0  gram  of  SO3  with  an  average  of  about  2.0  gram.  The  sulphuric 
acid  is  derived  but  to  a  slight  extent  ordinarily  from  ingested  sul- 
phates, being  mainly  dependent  on  the  sulphur  of  the  protein  ingested 
and  will  consequently  vary  widely  with  the  protein  content  of  the  diet. 
From  75  to  95  per  cent  of  the  total  sulphur  of  the  urine  is  ordinarily 
found  as  sulphate,  the  proportion  being  greatest  on  a  high  protein  diet. 
The  sulphate  excretion  is  increased  in  all  conditions  associated  with 
increased  decomposition  of  body  protein  as  in  acute  fevers  and  de- 
creased whenever  there  is  a  decrease  in  metabolic  activity. 

2.  Inorganic  Sulphates. — Folin's  Method. — Place  25  c.c.  of  urine  and  100 
c.c.  of  water  in  a  200-250  c.c.  Erlenmeyer  flask  and  acidify  the  diluted  urine 
with  10  c.c.  of  dilute  hydrochloric  acid  (i  volimie  of  concentrated  HCl  to  4  vol- 
umes of  water).  In  case  the  urine  is  dilute  50  c.c.  may  be  used  instead  of  25  c.c. 
and  the  volume  of  water  reduced  proportionately.  Add  10  c.c.  of  5  per  cent  bar- 
ium chloride  slowly,  drop  by  drop,  to  the  cold  solution  and  from  this  point  proceed 
as  indicated  in  the  method  for  the  determination  of  Total  Sulphates,  page  546. 

Calculate  the  quantity  of  inorganic  sulphates,  expressed  as  SO3,  in  the  twenty- 
four-hour  urine  specimen. 

Calculation. — Calculate  according  to  the  directions  given  imder  Total  Sul- 
phates, above. 

Interpretation. — On  an  average  about  90  per  cent  of  the  total  sul- 
phates of  the  urine  exists  as  inorganic  sulphates  but  the  proportion 
of  the  sulphates  existing  in  this  form  varies  widely,  being  greater  on 
a  high  protein  diet  than  on  a  very  low  protein  diet.  The  amount 
varies  with  the  total  sulphates  (which  see). 

3.  Ethereal  Sulphates. — Folin's  Method. — Principle. — The  inorganic  sul- 
phates are  removed  with  barium  chloride  and  the  conjugated  sulphates  then 
determined  after  hydrolysis. 

Procedure. — Place  125  c.c.  of  urine  in  an  Erlenmeyer  flask  of  suitable  size, 

^  It  is  considered  preferable  by  many  investigators  to  express  all  sulphur  valucs'in  terms- 
of  S  rather  than  SO3. 


548  PHYSIOLOGICAL    CHEMISTRY 

dilute  it  with  75  c.c.  of  water  and  acidify  the  mixture  with  30  c.c.  of  dilute  hydro- 
chloric acid  (i  volume  of  concentrated  HCl  to  4  volumes  of  water).  To  the  cold 
solution  add  20  c.c.  of  a  5  per  cent  solution  of  barium  chloride,  drop  by  drop.^ 
Allow  the  mixture  to  stand  about  one  hour,  then  filter  it  through  a  dry  filter  paper.' 
Collect  125  c.c.  of  the  filtrate  and  boil  it  gently  for  at  least  one-half  hour.  Cool 
the  solution,  filter  off  the  precipitate  of  BaS04,  wash,  dry  and  ignite  it  according  to 
the  directions  given  on  page  546. 

Calculation. — The  weight  of  the  BaS04  precipitate  should  be  multiplied  by 
2  since  only  one-half  (125  c.c.)  of  the  total  volume  (250  c.c.)  of  fluid  was  precipi- 
tated by  the  barivmi  chloride.  The  remaining  calculation  should  be  made  accord- 
ing to  directions  given  under  Total  Sulphates,  page  546. 

Calculate  the  quantity  of  ethereal  sulphates,  expressed  as  SO 3,  in  the  twenty- 
four-hour  urine  specimen. 

Interpretation. — The  excretion  of  ethereal  sulphates  (expressed  as 
SO3)  varies  ordinarily  from  o.i  to  0.25  gram  per  day  comprising  from 
5  to  15  per  cent  of  the  total  sulphur  excretion.  The  absolute  amount 
of  ethereal  sulphate  increases  with  increase  in  the  protein  of  the  diet  and 
particularly  with  increase  of  putrefactive  processes  in  the  intestine  or 
elsewhere.  The  amount  excreted  cannot  however  be  taken  as  an 
index  of  the  extent  of  intestinal  putrefaction. 

4.  Total  Svilphur.^ — ^Benedict's  Method.^ — Principle. — The  urine 
is  evaporated  and  ignited  with  a  solution  of  copper  nitrate  and 
potassium  chlorate.  Organic  matter  is  thus  destroyed  and  all  un- 
oxidized  sulphur  is  oxidized  to  the  sulphate  form  and  can  be  readily 
precipitated  with  barium  chloride  in  the  usual  manner.  The  method 
is  very  convenient  and  accurate. 

Ten  c.c.  of  urine  are  measiired  into  a  small  (7-8  cm.)  porcelain  evaporating  dish 
and  5  cc"  of  Benedict's  sulphur  reagent^  added.  The  contents  of  the  dish  are 
evaporated  over  a  free  flame  which  is  regulated  to  keep  the  solution  just  below  the 
boiling-point,  so  that  there  can  be  no  loss  through  spattering.  When  dryness  is 
reached  the  flame  is  raised  sUghtly  vmtil  the  entire  residue  has  blackened.    The 

^  See  note  (3)  at  the  bottom  of  p.  546. 

2  This  precipitate  consists  of  the  inorganic  sulphates.  If  it  is  desired,  this  BaSO* 
precipitate  may  be  collected  in  a  Gooch  crucible  or  on  an  ordinary  quantitative  filter 
paper  and  a  determination  of  inorganic  sulphates  made,  using  the  same  technic  as  that 
suggested 'above.  In  this  way  we  are  enabled  to  determine  the  inorganic  and  ethereal 
sulphates  in  the  same  sample  of  urine. 

^  Benedict:  Journal  of  Biological  Chemistry,  6,  363,  1909. 

*  If  the  urine  is  concentrated  the  quantity  should  be  slightly  increased. 

^  Crystallized  copper  nitrate,  sulphur-free  or  of  known  sulphur  content 200  grams. 

Sodium  or  potassium  chlorate 50  grams 

Distilled  water  to 1000  c.c. 

Denis  has  suggested  the  use  of  the  following  solution: 

Copper  nitrate 25  grams. 

Sodium  chloride 25  grams. 

Ammonium  nitrate 10  grams. 

Water  to  make 100  c.c. 

The  procedure  is  the  same  as  the  above  except  that  25  c.c.  of  urine  and  5  c.c.  of  reagent 
are  taken.     It  gives  accurate  results. 


URINE  549 

flame  is  then  turned  up  in  two  stages  to  the  full  heat  of  the  bxinsen  burner  and  the 
contents  of  the  dish  thus  heated  to  redness  for  ten  minutes  after  the  black  residue 
(which  first  fuses)  has  become  dry.  This  heating  is  to  decompose  the  last  traces  of 
nitrate  (and  chlorate).  The  flame  is  then  removed  and  the  dish  allowed  to  cool 
more  or  less  completely.  Ten  to  20  c.c.  of  dilute  (i  14)  hydrochloric  acid  is  then 
added  to  the  residue  in  the  dish,  which  is  then  warmed  gently  until  the  contents 
have  completely  dissolved  and  a  perfectly  clear,  sparkling  solution  is  obtained. 
This  dissolving  of  the  residue  requires  scarcely  two  minutes.  With  the  aid  of  a 
stirring  rod  the  solution  is  washed  into'  a  small  Erlenmeyer  flask,  diluted  with 
cold,  distilled  water  to  100-150  c.c,  10  c.c.  of  10  per  cent  barium  chloride  solution 
added  drop  by  drop,  and  the  solution  allowed  to  stand  for  about  an  hour.  It  is 
then  shaken  up  and  filtered  as  usual  through  a  weighed  Gooch  crucible.  Controls 
should  be  run  on  the  oxidizing  mixture. 

Calculation. — Make  the  calculation  according  to  directions  given  under  Total 
Sulphates,  page  546.  Calculate  the  quantity  of  sulphur  expressed  as  SO3  or  S, 
present  in  the  twenty-four-hour  urine  specimen. 

Interpretation. — The  total  sulphur  (SO3)  excretion  averages  about  2.5 
grams  per  day.  It  runs  more  or  less  parallel  with  the  decomposition 
of  endogenous  and  exogenous  protein  and  a  definite  ratio  between  the 
excretion  of  total  N  and  total  S  might  be  expected.  It  has  been 
suggested  that  the  ratio  5  :  i  expresses  this  relation  in  a  general  way  but 
no  constant  value  can  be  given.     Sec  Total  Sulj'jhates. 

5.  Total  Sulphur.  —  Osbome-Folin  Method. — Principle.  —  This 
method  depends  on  the  destruction  of  organic  matter  by  means  of 
sodium  peroxide.  It  is  employed  particularly  for  the  determination 
of  sulphur  in  foods  and  feces.  Benedict's  procedure  (see  above)  is 
simpler  and  fully  as  satisfactory  for  urine. 

Place  25  c.c.  of  urine-  in  a  200-250  c.c.  nickel  crucible  and  add  about  3  grams  of 
sodium  peroxide.  Evaporate  the  mixtiure  to  a  syrup  upon  a  steam  water-bath  and 
heat  it  carefully  over  an  alcohol  flame  xmtil  it  soUdifies  (15  minutes).  Now  remove 
the  crucible  from  the  flame  and  allow  it  to  cool.  Moisten  the  residue  with  1-2  c.c.  of 
water,  ^  sprinkle  about  7  8  grams  of  sodium  peroxide  over  the  contents  of  the  crucible 
and  fuse  the  mass  over  an  alcohol  flame  for  about  10  minutes.  Allow  the  crucible 
to  cool  for  a  few  minutes,  add  about  100  c.c.  of  water  to  the  contents  and  heat  at 
least  one-half  hour  over  an  alcohol  flame  to  dissolve  the  alkali  and  decompose  the 
sodiimi  peroxide.  Next  rinse  the  mixture  into  a  400  450  c.c.  Erlenmeyer  flask, 
by  means  of  hot  water,  and  dilute  it  to  about  250  c.c.  Heat  the  solution  nearly  to 
the  boihng-point  and  add  concentrated  hydrochloric  acid  slowly  until  the  nickelic 
oxide,  derived  from  the  crucible,  is  just  brought  into  solution.'  A  few  minutes' 
boiling  should  now  yield  a  clear  solution.  In  case  too  Uttle  peroxide  or  too  much 
water  was  added  for  the  final  fusion  a  clear  solution  will  not  be  obtained.  In  this 
event  cool  the  solution  and  remove  the  insoluble  matter  by  filtration. 

'  Sometimes  the  porcelain  glaze  cracks  during  heating,  in  whi.  1-  .  i-i-  th."  -..luiinn  should 
be  filtered  into  the  flask. 

*  If  the  urine  is  verj-  dilute  50  c.c.  may  be  used. 

^  This  moistening  of  the  residue  with  a  small  amount  of  waur  is  very  essential  and 
should  not  be  neglected. 

*  About  iS  c.c.  of  acid  are  required  for  S  grams  of  sodium  peroxide. 


550  PHYSIOLOGICAL    CHEMISTRY 

To  the  clear  solution  add  5  c.c.  of  very  dilute  alcohol  (about  18-20  per  cent)  and 
continue  the  boiling  for  a  few  minutes.  The  alcohol  is  added  to  remove  the 
chlorine  which  was  formed  when  the  solution  was  acidified.  Add  10  c.c.  of  a 
10  per  cent  solution  of  barium  chloride,  slowly,  drop  by  drop,^  to  the  liquid. 
Allow  the  precipitated  solution  to  stand  in  the  cold  two  days  and  then  filter  and 
continue  the  manipulation  according  to  the  directions  given  under  Total  Sul- 
phates, page  546. 

Calculation. — Make  the  calculation  according  to  directions  given  under  Total 
Sulphates,  page  546.  Calculate  the  quantity  of  sulphur,  expressed  as  SO3  or  S, 
present  in  the  twenty-four-hovir  urine  specimen. 

Interpretation. — See  page  549. 

(b)  Volumetric  Procedures 

6.  Volumetric  Determination  of  Ethereal  and  Inorganic  Sulphates. 
— ^Method  of  Rosenheim  and  Drummond.-— Pmzc?^/^.- — The  sulphates 
of  the  urine  are  precipitated  by  means  of  benzidine  solution,  the  pre- 
cipitate of  benzidine  sulphate  being  filtered  off  and  the  sulphuric 
acid  of  the  compound  titrated  with  N/io  KOH  using  phenolphthalein 
as  an  indicator.  This  is  possible  because  the  benzidine  is  a  very  weak 
base  and  its  sulphate  readily  dissociates.  It  is  necessary  that  excess 
of  HCl  be  avoided  in  the  precipitation  process. 

Procedure.^ — (a)  Inorganic  Sulphates. — Preparation  of  the  benzidine  solu- 
tion. Rub  4  grams  of  benzidine  (Kahlbaum)  into  a  fine  paste  with  about  10 
c.c.  of  water  and  transfer  to  a  2-hter  flask  with  the  aid  of  about  500  c.c.  of  water. 
Add  5  c.c.  of  concentrated  HCl  (sp.  gr.  1.19)  and  make  up  to  2  liters  with  distilled 
water.  One  hundred  and  fifty  c.c.  of  this  solution,  which  keeps  indefinitely, 
are  sufficient  to  precipitate  o.i  gram  H2SO4. 

Measure  25  c.c.  of  urine  into  a  250  c.c.  Erlenmeyer  flask  and  acidify  with 
dUute  hydrochloric  acid  (1:4)  until  the  reaction  is  distinctly  acid  to  Congo  red 
paper.  Usually  1-2  c.c.  of  dilute  acid  are  reqxiired.  One  himdred  c.c.  of  the 
benzidine  solution,  as  prepared  above,  are  then  run  in  and  the  precipitate, 
which  forms  in  a  few  seconds,  allowed  to  settle  for  ten  minutes.  Filter  with 
suction  and  wash  the  precipitate  with  10-20  c.c.  of  water  saturated  with  benzidine 
sulphate.  2  Transfer  the  precipitate  and  filter  paper  to  the  original  precipitation 
flask  with  about  50  c.c.  of  water  and  titrate  hot  with  N/io  KOH,  after  first 
adding  a  few  drops  of  saturated  alcohohc  solution  of  phenolphthalein. 

'  Calculation. — One  c.c.  of  N/io  KOH  corresponds  to  4.9  mg.  H0SO4  or  4.0  mg. 
of  SO3.  Multiply  the  number  of  cubic  centimeters  of  N/io  KOH  required  by 
4.9  and  by  4  to  get  the  amount  of  H2SO4  in  100  c.c.  of  the  urine  analyzed. 

^  See  note  (3)  at  the  bottom  of  p.  546. 

*  Rosenheim  and  Drummond:  Biochcm.  J.,  8,  143,  1914. 

'  In  order  to  obtain  accurate  results  it  is  most  important  that  the  precipitate  should  be 
finely  suspended  in  water  before  titration  and  this  again  entails  certain  precautions  during 
filtration  so  as  to  prevent  the  caking  together  of  the  precipitate.  The  authors  use  a  funnel 
of  6  cm.  diameter  and  a  perforated  porcelain  plate  (5-7  mm.)  covered  either  with  paper  pulp 
or  with  a  well-fitting  filter  paper.  Do  not  allow  the  precipitate  to  be  sucked  drj'  on  the  fil- 
ter.    The  final  filtrate  should  show  no  acid  reaction  to  Congo  red. 


URINE 


33- 


(b)  Total  Sulphates  (Inorganic  and  Ethereal). — Measure  25  c.c.  of  urine 
into  an  Erlenmeyer  flask,  add  2-2.5  c.c  of  dilute  HCl  ("1:41  and  20  c.c.  of  water 
and  boil  for  15-20  minutes.  The  ethereal  sulphates  are  hydrolized.'  Allow  the 
solution  to  cool  and  then  precipitate  the  sulphate  with  benzidine  as  in  the  deter- 
mination of  inorganic  sulphates.  The  titration  and  calculation  are  also  carried 
out  in  the  same  way. 

(c)  Ethereal  Sulphates. — Determine  the  total  sulphates  and  inorganic  sul- 
phates as  indicated  above.  Subtract  the  amount  of  inorganic  sulphate  from 
that  of  the  total  sulphate  and  obtain  the  amount  of  ethereal  sulphate  present. 

(d)  Total  Sulphur. — According  to  Rosenheim  and  Drummond-  the  benzidine 
method  may  be  employed  for  the  estimation  of  total  sulphur  in  the  solution  ob- 
tained on  the  oxidation  of  urine  by  the  Wolf-Osterberg^  modification  of  Bene- 
dict's method.  This  modification  involves  the  use  of  larger  quantities  of  urine 
than  the  Benedict  method  or  a  reduction  in  accuracy  and  hence  probably  has 
no  advantages  over  Benedict's  original  procedure.  See  below  for  modification 
of  Raiziss  and  Dubin. 

(e)  Neutral  Sulphur. — Neutral  sulphur  is  most  readily  determined  by  dif- 
ference. Subtract  from  the  total  sulphur  as  determined  by  one  of  the  methods 
given  above  the  amount  of  total  sulphates.  The  difference  corresponds  to  the 
neutral  sulphur  of  the  urine  sample  examined. 

Interpretation. — The  neutral  sulphur  of  the  urine  is  made  up  of 
cystine  and  related  bodies,  thiocyanate,  oxyproteic  acids,  etc.  It 
makes  up  ordinarily  from  5-25  per  cent  of  the  total  sulphur  of  the  urine, 
or  on  the  average  0.2  to  0.4  gram  per  day  calculated  as  SO3.  The 
absolute  amount  is  fairly  constant  for  a  given  individual  through  wide 
variations  of  protein  intake,  indicating  that  its  origin  is  mainly  en- 
dogenous, that  is,  that  it  arises  principally  from  the  decomposition  of 
tissue  protein.  On  this  account  the  percentage  of  the  total  sulphur 
excretion  existing  in  the  neutral  form  may  rise  to  25  per  cent  on  a  very 
low  protein  diet  and  decrease  to  5  per  cent  on  a  high  protein  diet,  the 
absolute  amount  remaining  nearly  constant.  In  fasting  percentages 
as  high  as  70  have  been  noted.  In  many  disorders  as  tuberculosis, 
cancer,  cystinuria,  etc.,  the  amount  may  be  relatively  and  in  some 
cases  absolutely  increased  but  no  fixed  relations  have  been  determined 
for  the  various  conditions. 

7.  Total  Sulphur. — Method  of  Raiziss  and  Dubin.  ^ — Principle. — The  urine  is 
oxidized  by  Benedict's  mclhod  (page  548)  the  sulphur  precipitated  as  benzidine 
sulphate  and  the  benzidine  titrated  with  N/io  permanganate  solution.  \'ery 
small  amounts  of  sulphur  may  be  determined  in  this  way. 

Procedure. — To  2  c.c.  of  urine  in  an  8  cm.  porcelain  dish,  add  0.5  c.c.  of  Bene- 
dict's reagent  (page  548)  and  evaporate  to  dryness  on  the  water-bath.     Heat  the  dish 

1  A  larger  amount  of  HCl  may  be  used  (20  c.c.  of  the  dilute  acid)  if  desired.  In  this 
case  it  is  necessary  to  neutralize  the  solution  carefully  after  boiling  and  again  add  dilute 
HCl  until  the  reaction  is  acid  to  Congo  red. 

^Rosenheim  and  Drummond:  Bioch.  Jour.,  8,  143,  1014. 

'  Wolf  and  Osterlierg:  Bioch.  Zcit.,  29,  429,  1910. 

*  Raiziss  and  Duliin:  Jour.  Biol.  Clicm.,  18,  297,  1914. 


552  PHYSIOLOGICAL   CHEMISTRY 

carefully  over  a  small  flame  till  the  contents  are  black,  and  then  heat  to  redness  for 
only  two  rm'nutes.  Cool,  add  2  c.c.  of  hydrochloric  acid  (1:4)  and  warm.  Neutralize 
the  clear  solution  with  NaOH  (10  per  cent)  and  again  acidify  with  i  drop  of  HCl 
(i  :4).  Add  25  c.c.  of  a  solution  of  benzidine  hydrochloride*  with  stirring.  Let 
stand  15  minutes  and  then  filter  off  on  an  asbestos  filter.  Transfer  the  precipitate 
to  the  filter  by  means  of  the  filtrate  and  wash  with  5  c.c.  of  cold  water,  drop  by  drop. 
Put  the  filter  into  a  500  c.c.  Erlenmeyer  flask,  add  i  c.c.  of  NaOH  (10  per  cent)  and 
200  c.c.  of  water.  Boil  the  suspension  for  five  minutes  and  then  cool  to  room  tem- 
perature. Add  20  c.c.  concentrated  sulphuric  acid  and  titrate  the  warm  solution  at 
once  with  N/io  potassium  permanganate  solution  till  a  distinct  pink  coloration  is 
obtained,  which  should  last  20  seconds.  In  titrating  at  first  add  only  about  0.5  c.c. 
at  a  time  and  toward  the  end  only  2  drops  at  a  time,  waiting  tiU  the  color  disappears 
before  further  addition  of  permanganate  solution.  As  the  titration  progresses  it 
win  be  noticed  that  the  yeUow  color  gradually  disappears,  the  solution  turning  prac- 
tically colorless.  It  is  at  this  stage  of  the  titration  that  care  should  be  taken  in  add- 
ing only  2  drops  of  permanganate  at  a  time. 

Calculation. — One  c.c.  of  N/io  potassium  permanganate  is  equivalent  to  0.099 
mg.  of  sulphur.  Multiply  the  number  of  cubic  centimeters  of  permanganate  used 
by  0.099  S'lid  divide  by  2  in  order  to  obtain  the  weight  of  sulphur  in  i  c.c.  of  urine. 
Calculate  the  day's  sulphur  output. 

Interpretation. — See  page  549. 

Phosphorus 

I.  Total  Phosphates. — Uranium  Acetate  Method. — Principle. — 
Standard  uranium  acetate  is  run  into  a  measured  quantity  of  urine 
until  all  of  the  phosphate  has  been  precipitated  as  insoluble  uranium 
phosphate.  An  excess  of  uranium  is  indicated  by  a  reddish  coloration 
with  potassium  ferrocyanide.  This  method  is  accurate  and  gives 
practically  the  total  phosphorus  of  urine  inasmuch  as  the  latter  exists 
generally  almost  entirely  as  phosphates. 

Procedvire. — To  50  c.c.  of  urine  in  a  small  beaker  or  Erlenmeyer  flask  add 
5  c.c.  of  a  special  sodium  acetate  solution^  and  heat  the  mixture  to  the  boiling- 
point.  From  a  burette,  run  into  the  hot  mixture,  drop  by  drop,  a  standard  solu- 
tion of  uranium  acetate^  until  a  precipitate  ceases  to  form  and  a  drop  of  the  mix- 
ture when  removed  by  means  of  a  glass  rod  and  brought  into  contact  with  a 

*  Six  and  seven-tenths  grams  of  benzidine  (Merck  reagent)  are  put  in  a  i-liter  flask,  29 
c.c.  of  hydrochloric  acid  (sp.  gr.  i.  12)  added  and  the  solution  diluted  up  to  the  mark. 

'  The  sodium  acetate  solution  is  prepared  by  dissolving  100  grams  of  sodium  acetate  in 
800  c.c.  of  distilled  water,  adding  100  c.c.  of  30  per  cent  acetic  acid  to  the  solution,  and 
making  the  volume  of  the  mixture  up  to  i  liter  with  water. 

'  Uranium  Acetate  Solution. — Dissolve  about  35.0  grams  of  uranium  acetate  in  i  liter 
of  water  with  the  aid  of  heat  and  3-4  c.c.  of  glacial  acetic  acid.  Let  stand  a  few  days 
and  filter.  Standardize  against  a  phosphate  solution  containing  0.005  gram  of  P2O6  per 
cubic  centimeter.  For  this  purpose  dissolve  14.721  grams  of  pure  air-dry  sodium  am- 
monium phosphate  (NaNH4HP04-l-4H20)  in  water  to  make  a  liter.  To  20  c.c.  of  this 
phosphate  solution  in  a  200  c.c.  beaker  add  30  c.c.  of  water  and  5  c.c.  of  sodium  acetate 
solution  (see  above)  and  titrate  with  the  uranium  solution  to  the  correct  end  reaction  as 
indicated  in  the  method  above.  If  exactly  20  c.c.  of  uranium  solution  are  required  i  c.c. 
of  the  solution  is  equivalent  to  0.005  gram  P2O8.  If  stronger  than  this  dilute  accordingly 
and  check  again  by  titration. 


URINE  553 

drop  of  a  solution  of  potassium  ferrocyanide  on  a  porcelain  test-tablet  produces 
instantaneously  a  brownish-red  coloration.^  Take  the  burette  reading  and 
calculate  the  P2O5  content  of  the  urine  under  examination. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uranium  acetate 
solution  used  by  0.005  to  determine  the  number  of  grams  of  P;:05  in  the  50  c.c. 
of  urine  used.  To  express  the  result  in  percentage  of  P^Oj  multiply  the  value 
just  obtained  by  2,  e.g.,  if  50  c.c.  of  urine  contained  0.074  gram  of  P-Os  it  would 
be  equivalent  to  0.148  per  cent. 

Calculate,  in  terms  of  P2O5,  the  total  phosphate  content  of  the  24- 
hour  urine  specimen. 

Interpretation. — The  excretion  of  phosphoric  acid  is  extremely 
variable  but  on  the  average  the  total  output  for  the  24  hours  is  about 
2.5  grams  expressed  as  PoOs-  Ordinarily  the  total  output  is  mainly  in 
the  form  of  phosphates  and  is  distributed  between  alkaline  and  earthy 
phosphates  in  the  ratio  of  2  :  i  but  this  is  likewise  inconstant.  The 
greater  part  of  the  phosphate  excretion  arises  from  the  ingested  food, 
either  from  the  preformed  phosphates  or  more  especially  from  the 
organic  combinations  as  phospho-  and  nucleoproteins.  The  ex- 
cretion is  consequently  very  largely  dependent  upon  the  phosphorus 
content  of  the  diet.  Some  of  the  phosphoric  acid  results  from  the 
breakdown  of  the  tissues  of  the  body,  and  this  endogenous  phosphoric 
acid  excretion  is  increased  in  conditions  of  increased  metabolism  as  in 
fevers.  The  findings  in  pathological  conditions  have  been  somewhat 
contradictory  due  to  lack  of  control  of  diet.  The  so-called  "phos- 
phaturias"  nearly  always  represent  decreased  acidity  and  not  in- 
creased phosphate  content  of  the  urine.  Such  conditions  are,  however, 
signiticant  as  indicating  a  possible  tendency  to  the  formation  of  phos- 
phatic  calculi. 

2.  Earthy  Phosphates. — Principle. — The  earthy  phosphates  are 
precipitated  by  making  the  urine  alkaline.  The  precipitate  is  filtered 
off,  dissolved  in  acid  and  titrated  with  uranium  acetate. 

Procedure. — To  100  c.c.  of  urine  in  a  beaker  add  an  excess  of  ammonium 
hydroxide  and  allow  the  mixture  to  stand  12  24  hours.  Under  these  conditions 
the  phosphoric  acid  in  combination  with  the  alkaUne  earths,  calcium  and  mag- 
nesium, is  precipitated  as  phosphates  of  these  metals.  Collect  the  precipitate 
on  a  filter  paper  and  wash  it  with  very  dilute  ammonium  hydroxide.  Pierce 
the  paper,  and  remove  the  precipitate  by  means  of  hot  water.  Bring  the  phos- 
phates into  solution  by  adding  a  small  amount  of  dilute  acetic  acid  to  the  warm 
solution.  Make  the  volume  up  to  50  c.c.  with  water,  add  5  c.c.  of  sodium  acetate 
solution,  and  determine  the  PjOj  content  of  the  mixture  according  to  the  direc- 
tions given  under  the  previous  method. 

Calculation. — Multiply  the  number  of  cubic  centimeters  of  uranium  acetate 
solution  used  by  0.005  to  determine  the  number  of  grams  of  PjOs  in  the  100  c.c. 

^  A  10  per  cent  solution  of  potassium  ferrocyanide  is  s;itisfactory. 


554  PHYSIOLOGICAL   CHEMISTRY 

of  iirine  used.  Since  loo  c.c.  of  urine  was  taken  this  value  also  expresses  the 
percentage  of  P2O5  present. 

Calculate  the  quantity  of  earthy  phosphates,  in  terms  of  P2O5,  present  in  the 
24-hour  urine  specimen. 

The  quantity  of  phosphoric  acid  present  in  combination  with  the  alkali 
metals  may  be  determined  by  subtracting  the  content  of  earthy  phosphates 
from  the  total  phosphates. 

Interpretation: — Ordinarily  the  earthy  phosphates  make  up  from 
30-40  per  cent  of  the  total  phosphate  excretion.  The  amount  varies 
with  the  excretion  of  calcium  and  magnesium  (which  see). 

3.  Total  Phosphorus. — {a)  Volumetric  Procedure. — Principle. — The 
organic  matter  is  destroyed  by  digestion  with  a  mixture  of  sulphuric 
and  nitric  acids  or  some  other  oxidizing  agent.  The  phosphorus  is  then 
precipitated  as  the  phosphomolybdate  and  determined  gravimetrically 
or  volumetricall3\ 

Preparation  of  the  Solution. — Pipette  10  c.c.  of  urine  (or  an  amount  of  sub- 
stance containing  about  20  mg.  of  P2O5)  into  a  Kjeldahl  flask.  Add  10  c.c.  of  a 
mixture  of  equal  parts  of  concentrated  H2SO4  and  concentrated  HNO3.  Digest 
over  a  low  flame  until  red  fumes  cease  to  come  off.  If  the  mixture  darkens  due 
to  the  charring  action  of  the  sulphuric  acid,  add  nitric  acid  from  a  separatory 
funnel  a  few  drops  at  a  time  and  continue  the  digestion.  When  the  mixture 
remains  clear  on  evaporation  to  the  point  where  white  sulphuric  fumes  come  off 
the  digestion  is  completed  by  heating  for  10-15  minutes  longer.  Cool  and  transfer 
the  solution  to  a  400  c.c.  Erlenmeyer  flask  with  the  aid  of  enough  water  to 
make  a  total  volxune  of  about  75  c.c.^ 

Instead  of  oxidizing  the  material  as  described  above  it  may  be 
ignited  with  magnesia  to  destroy  organic  matter.  About  2  grams  of 
the  solid  substance  or  25  c.c.  of  urine  (previously  evaporated  nearly 
to  dryness)  are  mixed  with  a  httle  more  than  an  equal  bulk  of  mag- 
nesium oxide  in  a  porcelain  dish  of  about  30  c.c.  capacity.  Five  c.c. 
of  magnesium  nitrate  solution  (see  Reagents  and  Solutions,  page  602) 
are  added  and  the  mixture  heated  very  gently  at  first,  then  gradually 
to  bright  redness.  The  mixture  is  cooled  and  transferred  with  water 
to  a  250  c.c.  flask.  An  excess  (20-30  c.c.)  of  HCl  are  added  and  the 
mixture  boiled  a  few  minutes.  Remove  from  the  flame  and  add  at 
once  enough  barium  chloride  solution  to  precipitate  any  sulphate 
present.     Cool,  make  to  mark,  filter  and  take  an  aliquot  for  analysis. 

Precipitation  of  the  Phosphomolybdate.^NeutraUze  the  solution  with 
ammonia,  make  slightly  acid  with  nitric  acid,  and  add  15  grams  of  ammonium 
nitrate  in  substance  (or  25  c.c.  of  a  60  per  cent  solution).  Heat  on  a  water-bath 
to  6o-65°C.  (not  higher)  and  add  30-40  c.c.  of  molybdate  solution,^  stir  and  let 

'  In  the  case  of  urine  it  is  possible  to  neutralize  this  acid  solution  with  ammonia,  make 
it  acid  with  acetic  acid  and  titrate  with  uranium  acetate  as  in  the  preceding  method. 

2  Made  by  adding  5  c.c.  of  concentrated  HNO3  to  100  c.c.  of  the  ordinary  molybdate 
solution  (see  Reagents  and  Solutions,  page  602). 


URINE  555 

stand  for  about  15  minutes  at  60-65°.  Filter  at  once  through  a  small  paper,' 
washing  the  precipitate  twice  by  decantation  with  2  per  cent  ammonium  nitrate 
solution  using  about  25  c.c.  each  time,  stirring  up  the  precipitate  well  in  each 
case,  and  allowing  to  settle.  Transfer  the  precipitate  to  the  filter  and  wash  with 
2  per  cent  ammonium  nitrate  solution  until  two  fillings  of  the  filter  (collected 
separately)  do  not  greatly  diminish  the  color  produced  with  phenolphthalein 
by  I  drop  of  the  standard  alkaU. 

Titration  of  the  Phosphomolybdate. — Transfer  the  precipitate  and  filter 
back  to  the  original  beaker  and  dissolve  in  a  small  excess  of  N  5  NaOH  Tabout 
2-3  c.c.  more  than  required  to  completely  dissolve  the  yellow  precipitate  .  Add 
about  100  c.c.  of  boiled  and  cooled  water  and  a  few  drops  of  phenolphthalein 
as  an  indicator  (a  red  color  should  be  observed  indicating  excess  of  NaOH) 
and  titrate  the  excess  of  NaOH  with  N/io  acid. 

Calculation. — Divide  the  number  of  cubic  centimeters  of  N/io  acid  required 
by  2  and  subtract  from  the  number  of  cubic  centimeters  of  N  5  NaOH  used. 
This  gives  the  number  of  cubic  centimeters  of  N/5  NaOH  required.  Multiply 
by  1.268  (the  equivalent  of  i  c.c.  of  N '5  NaOH  in  P2O5)  and  obtain  the  number 
of  milligrams  of  PX)i  in  10  c.c.  of  the  urine  analyzed.  Calculate  the  daily  output 
of  P2O5  in  this  case  from  the  24-hour  volume. 

Interpretation. — Nearly  all  of  the  phosphorus  of  the  urine  exists  as 
alkali  and  earthy  phosphates.  Consequently  the  total  phosphorus 
varies  in  the  same  way  as  the  total  phosphates  (which  see).  A  small 
portion  of  the  phosphorus  of  the  urine  may  exist  in  organic  com- 
bination though  never  in  a  reduced  form.  This  organically  bound 
phosphate  may  amount  to  from  1-4  per  cent  of  the  total  phosphorus 
excretion.  Little  is  known  with  regard  to  the  compounds  in  which  it 
occurs.  Possibly  some  glycerophosphoric  acid  may  occur  either  free 
or  as  lecithin. 

Gravimetric  Modification. — The  phosphorus  may  be  determined  somewhat 
more  accurately  by  substituting  a  gravimetric  procedure  for  the  above  titration. 
In  this  case  the  washed  phosphomolybdate  precipitate  is  dissolved  on  the  filter 
paper  with  ammonium  hydroxide  and  hot  water  to  make  a  volmne  of  not  more  than 
100  c.c.  Nearly  neutrahze  with  HCl,  cool,  and  add  about  10  c.c.  of  magnesia 
mixture  (see  Appendix)  from  a  burette.  Add  slowly  (about  i  drop  per  second) 
stirring  vigorously.  After  15  minutes  add  12  c.c.  of  ammoniimi  hydroxide 
solution  (sp.  gr.  0.90).  Let  stand  for  some  time  (two  hours  is  usually  enough) 
then  filter  and  wash  the  precipitate  with  2.5  per  cent  ammonia  until  practically 
free  from  chlorides.  Ignite  to  whiteness  or  to  a  grayish-white  ash  and  weigh. 
Multiply  the  weight  of  magnesium  pyrophosphate  thus  obtained  by  0.637  to  get 
the  weight  of  PjOs. 

Calculation.  -Calculate  as  explained  above. 

'  It  is  better  to  use  a  special  filter  tube  of  about  i  }-^  inches  diameter  (similar  to  a  Gooch 
filtering  tube)  in  which  is  placed  a  perforated  porcelain  plate  i '  s  inches  in  diameter,  covered 
with  a  layer  of  asbestos  1/8  inch  tliick.  Filtration  is  carried  out  with  suction  and  is  very 
rapid.  Ordinary  Gooch  crucibles  lined  with  asbestos  may  also  be  used  but  arc  not  so  s;itis- 
factory.  The  asbestos  used  should  be  specially  prepared  (see  Appendix).  For  a  good  dis- 
cussion of  the  details  of  procedure  and  sources  of  error  of  this  volumetric  method  sec 
Hibbard:  /.  Ind.  Eng.  Chein.,  5,  098,  1913. 


556  PHYSIOLOGICAL   CHEMISTRY 

Chlorides 

I.  Volhard -Arnold  Method. — Principle.— The  urine  is  acidified 
with  nitric  acid  and  the  chlorides  precipitated  with  a  measured  excess 
of  standard  silver  nitrate  solution.  The  silver  chloride  formed  is 
filtered  off  and  in  the  filtrate  the  excess  silver  nitrate  is  titrated 
back  with  standard  ammonium  thiocyanate  solution.  Ferric  am- 
monium sulphate  is  used  as  an  indicator.  A  red  color  due  to  the  forma- 
tion of  ferric  thiocyanate  indicates  that  an  excess  of  thiocyanate  is 
present  and  that  the  end  point  has  been  reached. 

Procedure. — Place  10  c.c.  of  urine  in  a  100  c.c.  voliimetric  flask,  add  20-30 
drops  of  nitric  acid  (sp.  gr.  1.2)  and  2  c.c.  of  a  cold  saturated  solution  of  ferric 
altim.  If  necessary,  at  this  point  a  few  drops  of  8  per  cent  solution  of  potassium 
permanganate  may  be  added  to  dissipate  the  red  color.  Now  slowly  run  in  a 
known  volimie  of  the  standard  silver  nitrate^  solution  (20  c.c.  is  ordinarily  used) 
in  order  to  precipitate  the  chlorine  and  insure  the  presence  of  an  excess  of  silver 
nitrate.  The  mixture  should  be  continually  shaken  during  the  addition  of  the 
standard  solution.  Allow  the  flask  to  stand  10  minutes,  then  fill  it  to  the  100 
c.c.  graduation  with  distilled  water  and  thoroughly  mix  the  contents.  Now 
filter  the  mixture  through  a  dry  filter  paper,  collect  50  c.c.  of  the  filtrate  and 
titrate  it  with  standardized  ammoniimi  thiocyanate  solution. ^  The  first  per- 
manent tinge  of  red-brown  indicates  the  end  point.  Take  the  burette  reading 
and  compute  the  weight  of  sodium  chloride  in  the  10  c.c.  of  urine  used. 

Calculation. — The  number  of  cubic  centimeters  of  ammonium  thiocyanate 
solution  used  indicates  the  excess  of  standard  silver  nitrate  solution  in  the 
50  c.c.  of  filtrate  titrated.  Multiply  this  reading  by  2,  inasmuch  as  only  one-half 
of  the  filtrate  was  employed,  and  subtract  this  product  from  the  number  of  cubic 
centimeters  of  silver  nitrate  (20  c.c.)  originally  used,  in  order  to  obtain  the  actual 
number  of  cubic  centimeters  of  silver  nitrate  utilized  in  the  precipitation  of  the 
chlorides  in  the  10  c.c.  of  urine  employed. 

To  obtain  the  weight  in  grams  of  the  sodium  chloride  in  the  10  c.c.  of  urine 
used,  multiply  the  number  of  cubic  centimeters  of  the  standard  silver  nitrate 
solution,  actually  utiUzed  in  the  precipitation,  by  o.oio.  If  it  is  desired  to  express 
the  result  in  percentage  of  sodium  chloride  move  the  decimal  point  one  place 
to  the  right. 

In  a  similar  manner  the  weight,  or  percentage  of  chlorine  may  be  computed 
using  the  factor  0.006  instead  of  0.0 10. 

^  Standard  silver  nitrate  solution  may  be  prepared  by  dissolving  29.042  grams  of  silver 
nitrate  in  i  liter  of  distilled  water.  Each  cubic  centimeter  of  this  solution  is  equivalent  to 
O.OIO  gram  of  sodium  chloride  or  to  0.006  gram  of  chlorine. 

'^This  solution  is  made  of  such  a  strength  that  i  c.c.  of  it  is  equal  to  i  c.c.  of  the  stand- 
ard silver  nitrate  solution  used.  To  prepare  the  solution  dissolve  13  grams  of  ammonium 
thiocyanate,  XHS4CN,  in  a  little  less  than  a  liter  of  water.  In  a  small  flask  place  20  c.c. 
of  the  standard  silver  nitrate  solution,  5  c.c.  of  the  ferric  alum  solution  and  4  c.c.  of  nitric 
acid  (sp.  gr.  1.2),  add  water  to  make  the  total  volume  100  c.c.  and  thoroughly  mix  the  con- 
tents of  the  flask.  Now  run  in  the  ammonium  thiocyanate  solution  from  a  burette  until  a 
permanent  red-brown  tinge  is  produced.  This  is  the  end-reaction  and  indicates  that  the 
last  trace  of  silver  nitrate  has  been  precipitated.  Take  the  burette  reading  and  calculate 
the  amount  of  water  necessary  to  use  in  diluting  the  ammonium  thiocyanate  in  order  that 
10  c.c.  of  this  solution  may  be  e.xactly  equal  to  10  c.c.  of  the  silver  nitrate  solution.  Make 
this  dilution  and  titrate  again  to  be  certain  that  the  solution  is  of  the  proper  strength. 


URINE  557 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the  24-hour  urine 
specimen. 

Interpretation. — From  10-15  grams  of  chlorine,  expressed  as  sodium 
chloride,  are  excreted  per  day,  on  the  average,  by  normal  adults.  The 
amount  is,  however,  closely  dependent  upon  the  chloride  content  of 
the  food  ingested.  In  fasting,  the  chloride  excretion  falls  rapidly  to 
a  very  minimal  quantity.  On  high  water  ingestion  it  is  increased. 
In  pneumonia  and  certain  other  acute  infectious  diseases  the  excretion 
of  chlorides  may  be  markedly  diminished  particularly  during  the 
periods  in  which  exudates  are  forming.  In  convalescence  and  with 
resolution  of  the  exudates  the  chlorine  excretion  rises  again.  A  de- 
crease has  also  been  noted  in  nephritis  associated  with  edema. 

2.  VoLhard -Harvey  Method.^ — Principle. — This  procedure  diflfers 
from  the  Volhard-Arnold  method  in  that  the  excess  of  silver  nitrate 
is  titrated  directly  without  filtering  and  hence  in  the  presence  of  the 
silver  chloride.  The  procedure  is  thus  more  rapid  but  the  exact  end 
point  is  more  difiicult  to  determine. 

Procedure. — Introduce  5  c.c.  of  urine  into  a  small  porcelain  evaporating 
dish  or  casserole  and  dilute  with  about  20  c.c.  of  distilled  water.  Precipitate 
the  chlorides  by  the  addition  of  10  c.c.  of  standard  silver  nitrate  solution-  and 
add  2  c.c.  of  acidified  indicator.^  Now  run  in  a  standard  ammonixun  thiocyanate 
solution^  from  a  burette  until  a  faint  red-brown  tint  is  visible  throughout  the 
mixture.  This  point  may  be  determined  readily  by  permitting  the  precipitate 
to  settle  somewhat.     Calculate  the  sodiimi  chloride  value  as  indicated  below. 

(If  a  red  tint  is  produced  when  the  first  drop  of  thiocyanate  is  added  an  addi- 
tional 10  c.c.  of  the  standard  silver  nitrate  solution  must  be  introduced.  The 
titration  should  then  proceed  as  above  described  and  proper  allowance  made  in 
the  calculation  for  the  extra  volimie  of  silver  nitrate  employed.) 

Calculation. — Since  2  c.c.  of  the  ammoniiun  thiocyanate  solution  is  equivalent 
to  I  c.c.  of  the  silver  nitrate  solution,  divide  the  burette  reading  by  2  and  sub- 
tract the  quotient  from  10  c.c,  the  quantity  of  silver  nitrate  solution  taken. 
This  value  is  the  niunber  of  cubic  centimeters  of  silver  nitrate  solution  actually 

1  Harvey:  Archives  of  Internal  Medicine,  6,  12,  19 10. 

2  See  p.  556. 

'This  is  prepared  as  follows:  To  30  c.c.  of  distilled  water  add  70  c.c.  of  33  per  cent 
nitric  acid  (sp.  gr.  1.2)  and  dissolve  100  grams  of  crystalline  ferric  ammonium  sulphate  in 
this  dilute  acid  solution.  Filter  and  use  the  filtrate  which  is  a  saturated  solution  of  the  iron 
salt.  This  single  reagent  takes  the  place  of  the  nitric  acid  and  ferric  alum  as  used  in  Vol- 
hard-Arnold method,  and  insures  the  use  of  the  proper  quantity  of  acid. 

*  This  is  a  solution  of  ammonium  thiocyanate  of  such  a  strength  that  2  c.c.  is  equivalent 
to  I  c.c.  of  the  silver  nitrate  solution.  First  make  a  concentrated  solution  by  dissolving  13 
grams  in  i  liter  of  water.  To  determine  the  requisite  dilution  to  make  such  a  solution  that 
2  c.c.  shall  be  equivalent  to  i  c.c.  of  the  silver  nitrate  solution  proceed  as  follows:  Introduce 
10  c.c.  of  the  silver  nitrate  solution  into  a  small  porcelain  evaporating  dish  or  casserole,  add 
30-50  c.c.  of  distilled  water,  2  c.c.  of  the  acid  indicator  and  titrate  as  described  above  with 
the  ammonium  thiocyanate  solution.  The  total  volume  of  the  concentrated  thiocyanate 
solution  excluding  that  used  in  this  titration  is  divided  by  10,  and  the  result  multiplied  by 
the  difiference  between  this  burette  reading  and  20  c.c.  This  will  give  the  volume  of  dis- 
tilled water  which  must  be  added  to  the  concentrated  thiocyanate  solution  to  render  2  c.c. 
equivalent  to  i  c.c.  of  the  silver  nitrate  solution. 


558  PHYSIOLOGICAL   CHEMISTRY 

used  in  the  precipitation  of  the  chlorides.  As  i  c.c.  of  the  silver  nitrate  solution 
is  equivalent  to  o.oi  gram  of  sodixim  chloride,  the  nxunber  of  cubic  centimeters 
of  silver  nitrate  solution  used  multiplied  by  o.oi  gram  will  give  the  weight  of 
sodium  chloride  in  the  5  c.c.  portion  of  urine  used.  The  weight  of  chlorine  may 
be  computed  by  using  the  factor  0.006  instead  of  o.oi. 

Calculate  the  weight  of  sodium  chloride  and  chlorine  in  the  24-hour  urine 
specimen. 

A  "short  cut"  method  of  calculating  the  24-hour  output  of  sodium  chloride 
consists  in  subtracting  the  burette  reading  from  20  c.c,  multiplying  this  value 
by  the  total  mine  volume  and  pointing  off  three  places. 

Interpretation. — See  above. 

3.  Dehn-Clark  Method^ — Principle. — In  this  method  any  organic  matter 
which  may  interfere  with  the  titrimetric  determination  of  the  chlorides  is  destroyed 
by  oxidation  with  sodium  peroxide.  The  chlorides  are  then  determined  by  the  Vol- 
hard-Arnold  titration  procedure. 

Procedure. — To  10  c.c.  of  urine  in  a  75-100  c.c.  casserole,  add  i. 0-1.2  grams  of 
sodium  peroxide  and  evaporate  the  mixture  to  dryness  on  a  boiling  water-bath.  In 
case  the  residue  is  not  pure  white,  thus  indicating  that  insufficient  sodium  peroxide 
has  been  added,  the  residue  should  be  moistened  with  distilled  water,  additional 
sodium  peroxide  added,  and  the  mixture  again  evaporated  to  dryness.  When  the 
oxidation  is  complete,  treat  the  mass  with  10-20  c.c.  of  distilled  water  and  stir 
untU  it  has  practically  all  been  brought  into  solution.  Then  introduce  a  bit  of  lit- 
mus paper  and  add  dilute  nitric  acid  (i:  i)  until  the  litmus  paper  turns  red  and  all 
efervescence  ceases.  Now  place  the  casserole  on  a  hot  plate  or  on  a  gauze  and  heat 
the  contents  almost  to  the  boiling-point.^  To  the  hot  solution  add  a  standard  solu- 
tion of  silver  nitrate  (see  page  556)  in  slight  excess.'  Filter  off  the  silver  chloride 
whUe  the  solution  is  still  hot  and  wash  the  precipitate  thoroughly  with  distilled 
water.  To  the  filtrate,  add  i  c.c.  of  a  saturated  solution  of  ferric  ammonium  sul- 
phate and  then  titrate  with  a  standard  solution  of  ammonium  thiocyanate  (see  page 
556)  untU  the  clear,  slightly  yellow  fluid  (or  the  opalescent,  milky  fluid,  in  case  there 
is  much  excess  of  silver  nitrate)  changes  to  a  slight  reddish-brown  color.  The  color 
of  the  end  point  varies  with  the  individual.  The  exact  end  point  reached  is  not  so 
important  as  is  the  securing  of  the  same  end  point  in  a  series  of  determinations  as 
that  obtained  in  the  standardization  of  the  standard  solutions  used. 

Calculation. — The  standard  solution  of  silver  nitrate  should  be  made  up  so  that. 
I  c.c.  equals  o.oio  gram  of  sodium  chloride  and  i  c.c.  of  the  ammonium  thiocyanate 
should  be  equivalent  to  i  c.c.  of  the  silver  nitrate  solution.  The  calculation  is  then 
exactly  the  same  as  in  the  Volhard-Arnold  method  (page  556)  except  that  the 
burette  reading  is  not  multiplied  by  2. 

Interpretation. — See  page  557. 

4.  Mohr's  Method. — Principle. — The  organic  matter  of  the  urine  is  destroyed 
by  ignition  with  potassium  nitrate.  To  the  solution  of  the  ash  potassium  chromate 
is  added  and  then  standard  silver  nitrate  solution  run  in  from  a  burette.     The  first 

'  Private  communication  to  the  author  from  Mr.  S.  C.  Clark. 

-  If  there  is  a  slight  precipitate,  due  to  silicic  acid  from  the  casserole,  this  is  filtered  ofif 
and  the  filtrate  collected  in  a  200  c.c.  beaker. 

3  This  point  is  most  easily  recognized  by  keeping  the  solution  hot  and  in  constant  agita- 
tion while  adding  the  silver  nitrate  so  that  the  silver  chloride  formed  coagulates  and  sinks, 
leaving  a  clear,  supernatant  fluid. 


URINE  559 

excess  of  silver  nitrate  over  that  necessary  to  precipitate  the  chlorides  reacts  with  the 
chromate  to  form  red  silver  chromatc  (Ag2Cr04).  This  indicates  the  end  point. 
The  method  is  accurate  when  applied  to  the  ash  of  urine  in  this  manner. 

Procedure. — To  lo  c.c.  of  urine  in  a  small  platinum  or  porcelain  crucible  or  dish 
add  about  2  grams  of  chlorine-free  potassium  nitrate  and  evaporate  to  dryness  at 
ioo°C.  (The  evaporation  may  be  conducted  over  a  low  flame  provided  care  is  taken 
to  prevent  loss  by  spurting.)  By  means  of  crucible  tongs  hold  the  crucible  or  dish 
over  a  free  flame  until  all  carbonaceous  matter  has  disappeared  and  the  fused  mass  is 
slightly  yellow  in  color.  Cool  the  residue  somewhat  and  bring  it  into  solution  in  a 
small  amount  (15-25  c.c.)  of  distilled  water  acidified  with  about  10  drops  of  nitric 
acid.  Transfer  the  solution  to  a  small  beaker,  being  sure  to  rinse  out  the  crucible  or 
dish  very  carefully.  Test  the  reaction  of  the  fluid,  and  if  not  already  acid  in  reac- 
tion to  litmus,  render  it  slightly  acid  with  nitric  acid.  Now  neutralize  the  solution 
by  the  addition  of  calcium  carbonate'  in  substance,  add  2-5  drops  of  neutral  potas- 
sium chromate  solution  to  the  mixture,  and  titrate  with  a  standard  silver  nitrate 
solution. 

This  standard  solution  should  be  run  in  from  a  burette,  stirring  the  liquid  in 
the  beaker  after  each  addition.  The  end-reaction  is  reached  when  the  yellow  color 
of  the  solution  changes  to  a  slight  orange-red.  At  this  point  take  the  burette  read- 
ing and  compute  the  percentage  of  chlorine  and  sodium  chloride  in  the  urine 
examined. 

Calculation. — The  calculation  is  made  exactly  as  in  the  Volhard-Arnold 
method,  page  556,  except  that  the  reading  is  not  multiplied  by  two. 

Calculate  the  quantity  of  sodium  chloride  and  chlorine  in  the  twenty-four- 
hour  urine  specimen. 

Interpretation. — See  page  557. 

Calcium  and  Magnesium 

McCrudden's  Methods.  ^^ — Principle. — Urine  contains  magnesium, 
phosphates  and  a  small  amount  of  iron,  each  of  which  will  interfere 
with  the  accurate  determination  of  its  calcium  content  if  proper  con- 
ditions of  acidity  are  not  maintained  during  the  precipitation.  In  the 
following  method  the  proper  acidity  is  attained  through  the  use  of 
sodium  acetate  and  hydrochloric  acid,  and  this  with  slow  addition  of 
the  ammonium  oxalate  reduces  the  danger  of  occlusion  of  magnesium 
oxalate,  calcium  phosphate,  or  ferric  phosphate  in  the  calcium  oxalate 
precipitate. 

The  calcium  oxalate  precipitate  is  either  ignited  and  weighed  as 
CaO  or  determined  volumetrically  by  titration  with  potassium  per- 
manganate. Magnesium  is  determined  in  the  filtrate  from  the  calcium 
determination  after  destruction  of  the  organic  matter.  It  is  determined 
in  the  usual  way  by  ignition  of  the  magnesium  ammonium  phosphate 
precipitate  and  weighing  as  the  pyrophosphate. 

'The  cessation  of  effervescence  and  the   presence   of  some  undecomposed  calcium 
carbonate  at  the  bottom  of  the  vessel  are  the  indications  of  neutralization. 
^McCrudden:  Jour.  Biol.  CItem.,  7,  83,  1910;   10,  187,  191 1. 


560  PHYSIOLOGICAL   CHEMISTRY 

Lyman  has  suggested  a  nephelometric  method  ioj:  the  determination 
•of  calcium  in  urine  and  feces. ^ 

Procedure  for  Calcitun. — ^If  the  urine  is  alkaline  make  it  neutral  or  slightly 
acid  and  filter.  Take  200  c.c.  of  the  filtered  urine  for  analysis.  If  it  is  only 
faintly  acid  to  Utmus  paper  add  10  drops  of  concentrated  hydrochloric  acid  (sp. 
gr.  1.20).  If  the  urine  is  strongly  acid  it  may  be  made  just  alkaUne  with  ammonia 
and  then  just  acid  with  hydrochloric  acid  after  which  the  10  drops  of  concentrated 
hydrochloric  acid  are  added.  Then  add  10  c.c.  of  2.5  per  cent  oxalic  acid.  Run 
in  slowly  with  stirring  8  c.c.  of  20  per  cent  sodiimi  acetate.  Allow  to  stand  over 
night  at  room  temperature  or  shake  vigorously  for  ten  minutes.  Filter  off  the 
precipitate  of  calcixun  oxalate  on  a  small  paper  and  wash  free  from  chlorides 
with  0.5  per  cent  ammonium  oxalate  solution.  The  precipitate  may  then  be 
dried,  ignited  to  constant  weight  and  weighed  as  calcium  oxide  or  it  may  be 
manipulated  voliunetrically  as  described  below. 

Volumetric  Procedure. — ^If  free  from  uric  acid  the  calcium  oxalate  precipitate 
may  be  washed  three  times  with  distilled  water,  filling  the  filter  about  two- 
thirds  full  and  allowing  it  to  drain  completely  before  adding  more.  A  hole  is 
made  in  the  paper  and  the  calcitmi  oxalate  washed  into  the  flask.  The  volmne 
of  the  fluid  is  brought  up  to  about  50  c.c.  and  10  c.c.  of  concentrated  sixlphuric 
acid  added.  Titrate  with  standard  potassimn  permanganate  solution  to  a  pink 
color  which  endures  for  at  least  a  minute. 

Calculation. — One  c.c.  of  N/io  permanganate  solution  is  equivalent  to  2.8 
mg.  of  CaO.     Calculate  the  daily  output  of  calciimi  expressed  as  CaO. 

Interpretation. — The  average  urinary  excretion  of  calcium  by  normal 
adults  lies  between  o.i  to  0.4  gram  (expressed  as  CaO)  per  day.  It  is 
dependent  very  largely  upon  the  amount  of  calcium  in  the  diet.  From 
10  to  40  per  cent  of  the  ingested  calcium  ordinarily  is  excreted  by  this 
channel,  the  greater  part  being  eliminated  by  the  feces.  The  pro- 
portion is  dependent  particularly  on  the  amount  of  calcium  in  the  food. 
If  the  calcium  ingestion  is  very  high  the  per  cent  of  the  total  excretion 
taking  place  by  way  of  the  kidneys  will  be  low,  and  vice  versa.  As  ex- 
cretion takes  place  by  way  of  the  intestine  as  well  as  by  the  kidneys  no 
conclusions  can  be  drawn  from  urinary  analyses  alone.  The  excretion 
of  calcium  may  be  greatly  increased  in  certain  bone  disorders  as  osteo- 
malacia. In  others  as  in  rickets  the  urinary  excretion  may  be  very 
low. 

Procedure  for  Magnesium. — Transfer  the  filtrate  from  the  determination 
of  calciimi  as  above  to  a  porcelain  dish,  add  about  20  c.c.  of  concentrated  nitric 
acid  and  evaporate  to  dryness.  Heat  the  residue  over  a  free  flame  until  the 
ammonium  salts  are  destroyed  and  the  residue  fuses.  After  cooUng  take  the 
residue  up  with  water  and  a  Uttle  hydrochloric  acid  and  filter  if  necessary. 
Dilute  to  about  80  c.c,  nearly  neutraUze  with  ammonia  and  cool.  Add  a  slight 
excess  of  sodium  acid  phosphate  and  then  ammonia  drop  by  drop  with  constant 
stirring  until  the  solution  is  alkaline  and  then  add  enough  more  slowly  with 

'Lyman:  Jour.  Biol.  Ckem.,  21,  551,  1915. 


URINE  561 

constant  stirring  to  make  the  solution  contain  one-fourth  its  bulk  of  dilute 
ammonia  fsp.  gr,  0.96).  Allow  to  stand  over  night.  Filter  and  wash  free  from 
chlorides  with  alcoholic  ammonia  solution  f  i  part  alcohol,  i  part  dilute  ammonia, 
3  parts  water).  The  precipitate  with  filter  paper  is  incinerated  slowly  and  care- 
fully with  good  supply  of  air  to  prevent  reduction,  in  the  usual  manner,  and  ignited 
and  weighed  as  the  pyrophosphate. 

Calculation. — To  obtain  the  weight  of  MgO  multiply  the  weight  of  magnesium 
pyrophosphate  by  0,3624. 

Interpretation. — The  daily  excretion  of  magnesium  by  way  of  the 
urine  usually  amounts  to  between  o.i  and  0,3  gram  (expressed  as  MgO). 
The  amount  depends  mainly  upon  the  diet.  Usually  50  per  cent  or 
more  of  the  excreted  magnesium  is  eliminated  by  the  kidneys,  the  re- 
mainder passing  out  in  the  feces.  The  proportion  varies,  however,  and 
it  is  impossible  to  draw  any  conclusions  from  the  urinary  output  alone. 
There  may  be  a  retention  of  magnesium  in  certain  bone  disorders  ac- 
companying a  loss  of  calcium;  in  osteomalacia  for  example.  Thus  the 
excretions  of  calcium  and  magnesium  do  not  necessarily  run  parallel. 

Determination  of  Calciiun  in  Ash  of  Foods  or  Feces. — Ignite  the  material  in  a 
crucible  to  a  white  ash  and  dissolve  the  ash  with  the  aid  of  a  little  hydrochloric  acid. 
Bring  the  volume  of  the  ash  solution  to  75-150  c.c.  INIake  just  alkaline  with  strong 
ammonia  added  drop  by  drop  (using  litmus  paper  or  alizarin  as  an  indicator).  Add 
concentrated  HCl  drop  by  drop  until  just  acid  to  litmus.  Then  add  10  drops  of 
concentrated  HCl  (sp.  gr.  1.20),  and  10  c.c.  of  2.5  per  cent  oxalic  acid.  Either 
of  two  procedures  may  then  be  followed,  (a)  The  solution  is  boiled  until  the  pre- 
cipitated calcium  oxalate  is  coarsely  crystalline,  and  then  an  excess  of  3  per  cent 
ammonium  oxalate  is  slowly  added  to  the  boiling  solution  and  the  boiling  continued 
until  the  precipitate  is  coarsely  crystalline.  (If  but  little  calcium  is  present  nothing 
will  precipitate  at  this  point  and  it  is  not  necessary  to  add  oxalate.)  Or  {b)  the 
flask  closed  with  a  rubber  stopper  is  shaken  vigorously  for  ten  minutes,  An  excess 
of  3  per  cent  ammonium  oxalate  is  then  added.  Cool  to  room  temperature.  Add 
8  c.c.  of  20  per  cent  sodium  acetate  solution.  (In  case  of  ash  of  feces  add  15  c.c.) 
The  solution  may  either  be  (c)  allowed  to  stand  over  night  or  {b)  stoppered  and 
vigorously  shaken  for  ten  minutes.  The  calcium  oxalate  is  filtered  off  on  a  small 
ash-free  paper  and  washed  free  from  chlorides  with  0.5  per  cent  ammonium  oxalate 
solution.  Either  of  two  procedures  may  next  be  followed,  (a)  The  precipitate 
and  filter  are  dried,  burned  in  a  platinum  or  porcelain  crucible  to  constant  weight  as 
CaO,  {b)  The  precipitate  is  washed  three  times  with  cold  distilled  water,  as  given 
under  the  method  for  urine  and  the  oxalate  titrated  with  potassium  permanganate. 

Magnesium  is  determined  in  the  filtrate  from  calcium  just  as  given  above. 

Sodium  and  Potassium 

Determination  of  Combined  Sodiimi  and  Potassium,     irum  ^c  tu  100  c.c.  of 

urine,  depending  upon  the  s()ocilk  gravity,  arc  oxidized  in  a  Kjeldahl  flask  with 

nitric  and  sulphuric  acids  as  in  the  Neumann  procedure  tor  total  phosphorus  (see 

page  554).     To  remove  the  sulphuric  acid  as  comnl'f'K    >-  i>ossiblc  transfer  with 

36 


62  PHYSIOLOGICAL    CHEMISTRY 


the  aid  of  a  little  water  to  a  platinum  dish  and  evaporate  to  drj^ness  over  a  free 
flame.  (The  alkalies  are  in  the  form  of  sulphate  and  do  not  volatilize.)  Dissolve 
the  residue  in  hot  water  with  the  aid  of  a  little  dilute  hydrochloric  acid.  Heat  to 
boiling  and  add  barium  chloride  solution  until  no  more  precipitate  forms.  While 
still  hot  add  an  excess  of  ammonia  and  ammonium  carbonate.  The  barium  chlor- 
ide precipitates  the  sulphates  and  part  of  the  phosphates:  the  ammonia  in  the 
presence  of  excess  barium  precipitates  the  rest  of  the  phosphates,  and  the  carbonate 
precipitates  the  calcium  and  most  of  the  magnesium,  as  well  as  the  excess  barium. 
Filter  and  wash  the  precipitate  well  with  hot  water  containing  a  few  drops  of  am- 
monia. Evaporate  filtrate  and  washings  to  dryness  and  heat  the  residue  to  dull 
redness  for  a  moment.  Redissolve  in  water  and  treat  again  with  ammonia  and 
ammonium  carbonate  to  remove  any  remaining  alkaline  earth  metals.  FUter  and 
wash  as  before.  Transfer  filtrate  and  washings  to  a  weighed  platinum  dish,  add  a 
few  drops  of  hydrochloric  acid  and  evaporate  to  dryness.  Heat  the  residue  gently 
to  remove  ammonium  salts  and  then  to  dull  redness  for  a  moment.  Desiccate 
and  weigh.  Reheat  to  constant  weight  which  represents  the  combined  chlorides 
of  sodium  and  potassium.  The  reagents  used  in  the  determination  must  be  tested 
and  found  free  from  alkali  metals  or  a  correction  made  for  the  alkali  metals  present 
in  the  reagents  used.  The  sodium  is  determined  by  difference  after  potassium  has 
been  estimated  by  the  method  given  below. 

Potassium, — Dissolve  the  alkaH  chlorides  from  the  preceding  determination  in  a 
little  water  and  add  a  slight  excess  of  lo  per  cent  platinic  chloride  over  that  neces- 
sary to  precipitate  all  of  the  alkali  present  calculated  as  sodium  chloride  (about  1 7 
c.c.  being  required  for  each  gram  of  sodium  chloride).  Evaporate  to  a  syrupy  con- 
sistencj-  on  the  water-bath  and  add  about  50  c.c.  of  80  per  cent  alcohol.  Stir 
occasionally  for  a  few  minutes.  This  operation  must  be  carried  out  in  the  absence  of 
ammonia  vapors.  Filter  through  a  weighed  Gooch  crucible,  washing  the  precipi- 
tate with  80  per  cent  alcohol  first  thoroughly  by  decantation  and  then  on  the  filter, 
for  some  time  after  the  filtrate  is  colorless.     Dry  at  iio-ii5°C.  and  weigh. 

Calculation. — ^Multiply  the  weight  of  potassium  platinic  chloride  by  0.1941  to 
obtain  the  amount  of  K2O  present.  Express  as  KCl  by  using  instead  of  this  factor 
the  factor  0.30712.  Subtract  from  the  \\  eight  of  total  alkali  chlorides  as  determined 
in  the  preceding  method,  the  weight  of  potassium  chloride  as  calculated  and  obtain 
the  amount  of  sodium  chloride  present. 

Interpretation. — The  average  alkali  excretion  of  an  adult  on  a  mixed  diet  is  about 
2-3  grams  of  potassium  expressed  as  KoO  and  4-6  grams  of  sodium  expressed  as 
Xa20.  The  ratio  of  Na  to  K  is  thus  about  5:3.  Both  the  ratio  and  the  absolute 
amounts  of  these  elements  excreted  are,  however,  largel}'  dependent  upon  the  salt 
content  of  the  diet.  Because  of  the  non-ingestion  of  sodium  chloride  and  the 
accompanying  destruction  of  potassium-containing  body  tissues,  the  urine  during 
fasting  contains  more  potassium  than  sodium  salts.  The  excretion  of  the  bases, 
particularly  K,  maj'  be  increased  in  fevers  and  in  acidosis. 

Iron 

Method  ofWolter.' — Principle. — The  urine  is  ashed,  the  ash  dissolved,  and  the 
iron  present  oxidized  to  the  ferric  form  by  means  of  hydrogen  peroxide.  The  iron  is 
then  determined  iodometrically. 

'  Wolter:  Bioch.  Zeil.,  24,  108.  iqio. 


URINE  563 

Procedure. — The  24-hour  specimen  of  urine  is  treated  with  30  c.c.  of  concen- 
trated iron-free  nitric  acid  and  then  evaporated  to  low  volume  in  a  large  evaporating 
dish  on  the  water-bath.  Transfer  to  a  small  evaporating  dish.  Heat  to  dryness  on 
the  sand  bath  and  then  char,  using  a  small  flame.  Transfer  the  charred  mass  by 
means  of  a  glass  spatula  to  a  crucible.  The  remaining  material  in  the  evaporating 
dish  is  transferred  with  the  aid  of  a  little  hot  water  and  a  rubber  "policeman"  to  a 
second  crucible.  Evaporate  to  dryness  on  the  water-bath  and  then  ash  the  material 
in  both  crucibles.  Dissolve  the  ash  in  about  30  c.c.  of  iron-free  hydrochloric  acid, 
transfer  to  an  Erlenmeyer  flask,  add  2  c.c.  of  hydrogen  peroxide  and  boil  for  three- 
quarters  of  an  hour.  After  cooling,  2  grams  of  potassium  iodide  and  a  few  drops  of 
fresh  starch  paste  are  added.  The  liberated  iodine  is  titrated  with  X/ioo  thiosul- 
phate  solution.  Controls  should  be  run  on  reagents.  A  correction  of  0.32  mg.  is 
usually  necessary  for  the  undecomposed  hydrogen  peroxide.  The  thiosulphate  solu- 
tion is  made  up  as  needed  from  an  N/io  stock  solution  by  dilution.  It  is  standard- 
ized against  an  iron  solution  containing  2  mg.  of  iron  in  10  c.c.  The  number  of 
cubic  centimeters  of  thiosulphate  used  in  titration  of  the  iodine  set  free  from  the 
ash  solution  is  multiplied  by  the  iron  equivalent  of  i  c.c.  of  the  thiosulphate  (about 
0.2  mg.)  to  obtain  the  total  amount  of  iron  in  the  24-hour  specimen  of  urine.  From 
1-5  mg.  of  iron  are  usually  excreted  per  day. 


CHAPTER  XXVII 
METABOLISM 

Metabolism  is  a  part  of  that  complex  series  of  processes  grouped 
together  under  the  head  of  Nutrition.  It  embraces  a  consideration 
of  those  changes  taking  place  in  the  body  other  than  those  customarily 
classified  as  secretion,  digestion,  excretion,  etc.  Metabolism  may  be  de- 
fined as  all  chemical  and  physical  changes  which  occur  in  living  matter 
and  which  constitute  the  basis  of  the  material  phenomena  of  life.  This 
conception  of  metabolism  holds  for  the  simple  individual  cell  of  the 
amoeba  as  well  as  for  the  complex  mechanism  of  the  human  body.  There 
are  two  types  of  metabolism,  one  constructive,  the  other  destructive. 
The  constructive  metabolism  is  termed  anabolism;  the  destructive 
metabolism  is  termed  catabolism. 
Thus: 

,,  ^   ,   ,.  (  Anabolism  (constructive  metabolism). 

Metahohsm        „      ,   ,.       ,,  .  ■,    t      n 

[  Lataboksm  (destructive  metabolism). 

In  general  we  may  say  that  the  main  bulk  of  the  food-stuffs  of  the  diet, 
i.e.,  protein,  fat  and  carbohydrate,  is  transformed  in  the  gastro-in- 
testinal  tract  and  that  the  end-products  of  this  transformation  are 
carried  to  the  cells  of  the  body  and  there  built  up  by  anabolic  (synthetic) 
processes  into  cell  structure  or  stored  as  a  reserve  to  be  used  as  required. 
All  living  cells  undergo  wear  and  tear  in  the  course  of  their  life  cycle. 
By  catabolic  (cleavage)  processes  therefore  a  portion  of  the  living  cell 
substance  or  of  the  stored  material  is  reduced  to  simpler  fragments  and 
these  are  eliminated  from  the  body  after  having  yielded  the  bulk  of 
their  energy  in  the  form  of  heat  or  mechanical  work.  It  is  apparent, 
therefore,  that  the  chemical  side  of  metabolism  is  closely  associated 
with  the  physical  side.  Each  of  the  three  types  of  food-stuffs  (protein, 
fat  and  carbohydrate)  is  concerned  with  the  upkeep  of  the  tissues  and 
with  the  liberation  of  energy.  It  is  true,  however,  that  the  main 
burden  of  the  upkeep  falls  upon  the  proteins  whereas  the  combustion  of 
fats  and  carbohydrates  yields  the  major  portion  of  the  required  energy. 
The  above  facts  are  embraced  in  the  following  scheme: 

564 


METABOLISM  565 

THE  CELL 

PROTEIN.    FAT,       1  ,  ^^,^  t,.,^^,.^    „ 

CARBOHYDRATE     /    ^  ^  END-PRODUCTS 


COMBUSTION 

Without  doubt  both  anabolic  and  catabolic  processes  are  going  on  in- 
cessantly within  every  individual  living  cell.  At  one  time  the  anabolic 
phase  will  be  more  prominent;  at  another  the  catabolic  activity  will  be 
in  the  ascendency.  It  should  also  be  borne  in  mind  that  metahoUsm 
implies  a  transfonnation  of  energy  as  well  as  an  exchange  of  materials. 

For  further  brief  discussions  of  certain  phases  of  metabolism  see 
the  following  experiments.  A  detailed  discussion  being  out  of  place 
in  this  volume,  the  reader  is  referred  to  the  following  books : 

(i)  Taylor's  "Digestion  and  ]\Ietabolism, "  Lea  and  Febiger,  191 2. 

(2)  Sherman's  "  Chemistry  of  Food  and  Nutrition,"  Macmillan. 

(3)  Osier  &  McCrae's  "Modern  Medicine,"  Vol.  II,  Second  Edi- 
tion, 1914,  Lea  and  Febiger.  The  author's  section  on  "General  Con- 
sideration of  ^Metabolism, "  pages  549-673. 

METABOLISM  EXPERIMENTS 

I .  Metabolism  Procedures  Involving  the  Manipulation  of  the  Urine 

1.  Collection  and  Preservation  of  the  Urine. — In  metabolism  tests, 
such  as  those  given  in  this  chapter  the  accurate  collection  of  the  urine 
for  the  exact  24-hour  period  is  of  the  utmost  importance.  Proceed  as 
follows:  Empty  the  bladder  at  a  given  hour,  e.g.,  8  a.m.  and  discard  tJie 
urine.  Prepare  a  thoroughly  clean  bottle  of  proper  size,  introduce  into 
it  sufficient  toluene  to  cover  the  bottom  of  the  bottle  and  use  this  bottle 
for  the  collection  of  all  urine  voided  from  8  a.m.  until  8  a.m.  the  next 
day.  During  the  day,  when  not  actually  in  use  for  the  introduction 
of  a  urine  fraction,  the  bottle  should  be  kept  in  a  refrigerator  or  cold 
room  in  order  that  the  sample  may  not  deteriorate  before  it  is  examined. 
Measure  the  volurne  of  the  sample  and  determine  its  specific  gravity 
(see  Chapter  XXI)  and  reaction  before  proceeding  to  the  quantitative 
estimation  of  any  specific  urinary  constituents. 

2.  Complete  Analysis  of  Urine. — Ingest  an  ordinary  mixed  diet  (or  any  special 
diet)  and  collect  the  urine  accurately  for  a  24-hour  period  (see  above).  Measure 
the  volume  of  the  sample,  determine  the  specific  gravity  and  preserve  the  urine 
(see  above)  until  the  following  constituents  have  been  determined  (for  Methods 


566 


PHYSIOLOGICAL   CHEMISTRY 


of  Analysis,  see  Chapter  XXVI).  Total  solids,  titratable  acidity,  hydrogen  ion 
concentration,  total  nitrogen,  amino-acid  nitrogen,  ammonia,  urea,  uric  acid, 
creatinine,  total  sulphur,  ethereal  sulphates,  inorganic  sulphates,  neutral  sulphur 
(by  difference)  total  phosphates  and  sodiiun  chloride. 

Calculate  the  nitrogen  and  sulphur  "partitions,"  i.e.,  the  percentage  of  the 
total  nitrogen  and  sulphur  which  occur  in  the  different  forms  and  tabulate  the  data 
from  the  complete  analysis.  Compare  your  results  with  those  listed  in  the  table 
on  pages  371  and  578. 

3.  Hyperglycemia   Produced   by    Carbohydrate  Ingestion.^The 

average  glucose  content  of  normal  blood  is  somewhat  less  than  o.i  per 
cent.  This  is  increased  (hyperglycemia)  on  the  ingestion  of  carbo- 
hydrate food.     The  increase  is  noted  more  quickly  after  the  ingestion  of 


0.18 

T' 

\ 

"^ 

A 

\ 

a 

II 

\ 

\ 

4 

1 
1 
1 

\ 

\ 
\ 

_B__ 

\ 

^ 

1/ 

V'^x 

_C-- 

il 

n 

y' 

\ 
\ 

\ 

^--^ 

-^^^ 

om 

^V,s. 

^        \ 

iKr--^ 

"^^ 

^\ 

Vz 


Fig.  169. 


J'/z 


Z        ZVz      3 
Hours 


3Vz 


^Vz     5 


-Blood  Sugar  as  Influenced  by  Diet. 
A  =  glucose;  B  —  starch; 
C  =  starch  and  fat;  D  =  fat. 


monosaccharides  than  after  the  ingestion  of  the  more  complex  carbo- 
hydrates. After  the  ingestion  of  100  grams  of  glucose  or  starch  an 
increase  in  the  sugar  of  the  blood  sometimes  occurs  in  five  minutes.^ 
(See  Fig.  169.) 

(a)  Influence  of  Glu(iose. — In  the  morning  before  breakfast,  or  three  to  five 
hours  after  breakfast,  determine  the  normal  sugar  content  of  your  blood  by  means 
of  some  accurate  micromethod.  (See  Chapter  XVI.)  Ingest  100  grams  of 
glucose  dissolved  in  250  c.c.  of  water,  and  again  determine  the  blood  sugar 
level  at  intervals  of  5,  15  and  30  minutes  and  one,  two  and  three  hoiirs.  (Plot 
a  curve  similar  to  the  one  shown  in  Fig.  169.  The  urine  may  also  be  examined 
for  sugar  at  intervals  of  one  hour  after  the  sugar  ingestion. 

Repeat  the  experiment  on  another  day  using  250  grams  of  glucose  and  com- 
pare the  results  with  those  obtained  after  the  ingestion  of  100  grams.  Explain 
your  findings.  If  desired  this  experiment  may  be  combined  with  the  ones  on 
"Alimentary  Glycosuria,"  and  "  Carbohydrate  in  Feces,"  see  pages  568  and  591. 


1  Jacobson:  Bioch.  Zeit.,  56,  471,  1913. 


METABOLISM  567 

(b)  Influence  of  Starch. — Repeat  the  experiment  as  given  above  for  glucose 
except  that  170  grams  of  white  bread  or  100  grams  of  starch  made  into  a  paste- 
are  substituted  for  the  100  grams  of  glucose. 

The  experiment  may  be  repeated  as  described  above  using  an  increased 
amount  of  starch. 

The  various  experiments  may  be  conducted  on  patients  suffering  from  dia- 
betes meUitus  if  such  are  available  and  instructive  data  collected.  The  ahmen- 
tary  hyperglycemia  will  generally  be  more  prolonged  than  in  the  case  of  normal 
subjects. 

4.  Influence  of  Physical  Exercise  upon  Blood  Sugar. — After  strenuous  physical 
exertion  by  a  normally  nourished  individual  there  is  an  increase  in  the  sugar  concen- 
tration of  the  blood. ^  Similar  increases  are  not  shown  by  resting  individuals  simi- 
larly nourished  nor  by  fasting  individuals  after  strenuous  physical  exercise.  This 
point  is  illustrated  in  the  following  protocol: 

INFLUENCE  OF  PHYSICAL  EXERCISE  OX  BLOOD  SUGAR  (Normal  Man) 

'  Blood  Blood 

Day  Experimental  conditions  examined,         sugar, 

hour  per  cent 


Normal. 

7  A.  M. 

0.043 

Marched  8  miles  in  2  hours  ;  ate  200  grams  sucrose. 

12  A.M. 

0  080 

Normal.                                                                           1 

7  A.  M.   ' 

0.045 

Complete  rest;  ate  200  grams  sucrose. 

12  A.M. 

0  055 

1  Fasting  and  complete  rest. 

7  A.  M. 

0.047 

1 
Fasting  and  complete  rest. 

12  A.M. 

0.045 

Fasting. 

7  A.  M. 

0.047 

Fasting  and  2 -hour  march  (8  miles).  '   12  A.  M.  ,      0.053 


The  increase  in  blood  sugar  under  the  influence  of  exercise  occurs  rather  sooner  in 
the  diabetic  organism.     Typical  data  follow: 

Experiment. — Ingest  a  simple  uniform  diet  (see  Experiment  34,  page  592)  for 
five  days  taking  the  first  meal  after  12  o'clock  (noon)  and  the  last  one  before  10 
P.  M.  On  the  morning  of  the  second  day  (7  .\.  M.)  determine  the  sugar  in  your 
blood  (see  methods,  Chapter  X\T).  About  three  hours  later  take  a  brisk  walk 
for  8  miles  covering  the  distance  in  about  two  hours  and  consume  200  grams 
sucrose  during  the  walk.  Make  a  second  analysis  of  the  blood  sugar.  On  the  third 
day  analyze  your  blood  for  sugar  at  7  A.  M.  and  again  at  noon,  remaining  quiet  in 
the  meantime.  The  fourth  day  should  be  passed  without  physical  exertion  whereas 
on  the  second  day  between  10  A.  M.  and  12  M.  a  brisk  S-mile  walk  is  taken  but  no 

'  In  making  starch  paste,  rub  up  the  dry  starch  in  a  mortar  wiiJi  cold  water  and  pour  the 
suspended  starch  granules  into  boiling  water  and  stir. 
*  Moraczewski:  Bioch.  Zeit.,  71,  268,  1915. 


568  PHYSIOLOGICAL   CHEMISTRY 

sucrose  ingested.     Sugar  analyses  should  be  made  at  7  A.  M.  and  12  M.  each  day. 
INFLUENCE  OF  EXERCISE  ON  BLOOD  SUGAR  (Diabetes  Patient) 

Blood 

Blood 

.^^^^•^       su^'ar  . 

Day  examined,       °     '  Experimental  conditions 


hour 


per 
cent 


I  I 

8  A.  M.     0.062  j  Rest.     Diet  consisting  of  2500  grams  milk,  300  grams 
I    bread,  50  grams  fat. 

8  A.  M.     0. 120    Eight-mile  march  (2  hours).     Diet  as  above. 


3  P.  M.     0.08s 


8  A.  M.     0.055  t  Rest.     Diet  as  on  first  day. 


3  P.  M. 

0.050 

Rest.     Diet  as  on  first  day. 

8  A.  M. 

0.050 

Rest.     Diet  as  on  first  day. 

4 

3  P.  M. 

O.OS5 

Rest.     Diet  as  on  first  day. 

5 

8  A.  M. 

1 

0.084 

Eight-mile  march  (2  hours). 

Diet  as  on  first  day. 

Calculate  your  results,  tabulate  them  and  compare  them  with  those  given  above. 

5.  Alimentary  Glycosuria. — Normal  urine  contains  a  trace  of  glucose  but  not 
enough  to  permit  detection  by  the  ordinary  tests  used  in  urinary  analysis.  If 
more  glucose  is  ingested  than  can  be  absorbed  and  assimilated  by  the  body  the 
excess  will  be  eliminated  in  the  iirine.  The  "assimilation  limit"  for  the  glucose 
has  been  exceeded,  and  a  transient  alimentary  glycosuria  results.  To  demon- 
strate this,  glycosuria  proceed  as  follows :  Before  breakfast  or  luncheon  empty 
the  bladder  and  test  the  urine  for  sugar  by  any  reliable  test  (see  Chapter  XXXII). 
If  the  test  is  negative,  ingest  along  with  the  other  articles  of  diet,  250  grams  of 
glucose,  sucrose,  or  lactose  dissolved  in  water.  Empty  the  bladder  at  the  end 
of  every  hour  for  a  period  of  three  hours  and  test  the  urine  for  reducing  sugar 
and  the  sugar  ingested. 

Was  there  any  glycosuria  and  if  so  how  soon  after  the  sugar  ingestion  did  it 
appear?  If  no  glycosuria  resulted  repeat  the  test  on  a  subsequent  day  using 
a  larger  quantity  of  sugar.  If  desired,  the  sugar  in  the  urine  may  be 
determined  quantitatively  by  one  of  the  methods  given  in  Chapter  XXVI. 

This  experiment  may  be  made  more  complete  by  making  determinations  of 
blood  sugar  at  short  intervals  as  described  in  Experiment  3,  page  566.  If 
desired,  data  on  glycosuria,  hyperglycemia  and  carbohydrate  in  feces  (page  591) 
may  be  collected  from  one  experiment. 

6.  Absorption  of  Carbohydrate  as  Influenced  by  Fat  Ingestion. — When  fat  is 
eaten  along  with  carbohydrate  food  the  absorption  of  the  latter  is  somewhat 
delayed.  This  has  been  shown  experimentally.^  To  demonstrate  the  point  pro- 
ceed as  follows:  Determine  the  content  of  sugar  in  the  blood  at  various  intervals 

^  Jacobson:  Bioch.  Zeit.,  56,  471,  1913. 


METABOLISM  569 

after  the  ingestion  of  170  grams  of  white  bread  as  described  in  Experiment  3(b), 
page  567.  Plot  a  curve  for  these  values  similar  to  the  one  shown  in  Fig.  169,  page 
566.  On  a  later  day  repeat  the  experiment  and  ingest  1 70  grams  of  white  bread  and 
85  grams  of  butter.  Plot  the  curve  for  these  blood  sugar  concentrations  along  with 
blood  sugar  values  obtained  after  the  ingestion  of  white  bread  as  described  above. 
Has  the  fat  exerted  any  influence  upon  the  absorption  of  the  carbohydrate?  Re- 
peat the  above  experiment  on  a  case  of  diabetes  mellitus  if  such  is  available  and 
note  that  fat  exerts  the  same  influence  upon  carbohydrate  absorption  as  it  exerts  in 
the  normal  human  bod)'. 

7.  Time  Relations  of  Protein  Metabolism. — It  is   a  well-known 

physiological  fact  that  an  interval  elapses  between  the  ingestion  of  protein 
food  and  the  appearance  in  the  urine  of  certain  products  representing  the 
complete  catabolism  of  this  food.  For  example,  if  one  ingests  an  excess 
of  protein  material  an  interval  elapses  before  the  urine  gives  evidence 
of  the  complete  excretion  of  certain  products  representative  of  the 
catabolism  of  the  protein.  Urea  is  the  chief  of  these.  The  term  "nitro- 
gen lag"  has  been  used  to  designate  the  period  elapsing  between  the 
ingestion  of  protein  and  the  excretion  in  the  urine  of  a  quantity  of 
nitrogen  equivalent  to  that  contained  in  the  protein. 

Experiment. — Ingest  a  simple  uniform  diet  whose  exact  composition  has  been 
determined  by  analysis  or  whose  approximate  composition  has  been  estimated. 
(See  table  below.)  Continue  this  diet  from  one  to  four  days.  Collect  the 
urine  in  two-hour  periods  from  7  A.  M.  to  1 1  P.  M.  and  in  an  eight-hour  period  be- 
tween II  P.  M.  and  7  A.  M.  Analyze  each  specimen  for  total  nitrogen  or  urea. 
At  the  end  of  this  preliminary  period  add  to  the  uniform  diet,  at  one  meal,  a 
weighed  quantity  (150-250  grams)  of  lean  meat  specially  prepared  and  analyzed. 
Collect  the  urine  in  periods  as  before  and  determine  total  nitrogen  or  urea. 
Calculate  the  total  nitrogen  or  urea  excretion;  tabulate  the  data  and  plot  curves 
showing  the  course  of  the  nitrogen  excretion  on  the  various  days  of  the  experi- 
ment.    How  long  was  the  "nitrogen  lag?" 

COMPOSITION'  OF  COMMON  FOODS' 


Food 

1  Water 

Protein 
(NX6.2S) 

Fat 

'  Carbo- 
hydrates 

Ash 

Calories  per 
pound 

Beef 

Loin 
Ribs 
Round 

Percent 
61.3 
57. 0 
67.8 

Per  cent 
19.0 
17.8 
30.9 

Per  cent 
19.1 
34.6 
10.6 

Per  cent 

Per  cent 
i.o 
0.9 
1. 1 

II3S 

1338 

8l3 

Mutton 

Loin 

Leg 

Shoulder 

SO.  2 
67.4 
67.2 

16.0 
19-8 
19-5 

33.1 

13.4 
13.9 

0.8 
1. 1 
1.0 

1643 
86s 
905 

Sweetbreads 

1      70.9 

16.8 

13. I 

1.6 

800 

Liver 

1     6S.6 

30.3 

3.1 

3.S 

1.3 

539 

Heart 

62.6 

16.0 

30.4 

1.0 

1135 

Tongue 

70.8 

18.9 

9.3 

1.0 

719 

Brain 

80.6 

8.8 

9.3 

1. 1 

540 

Ham 

49.3 

33.5 

31.0 

5.8 

1366 

1  Sherman's  "Food  Products,"  Macmillan,  1914. 


570 


PHYSIOLOGICAL   CHEMISTRY 


Food                                Water      ^Sx'&)        ^^*       h?dra°^       Ash 

Calories  per 
pound 

i 

;  Per  cent 

r-i,;„i,»„                     Broilers                     i      74.8 
Chicken                     p^^i                           1      63.7 

Per  cent      Per  cent 
21.5               2.5 
19.3       1      16.3 

Per  cent 

Per  cent 
I.I 
i.o 

492 
1016 

„..                            Halibut                         75.4            18.6              5.2 
^'^•^                            Salmon                           64.6             22.0             12.8 

i.o 

1-4 

550 
922 

Oatmeal  (boiled)         84.5               2.8              0.5 

Cereals                     Rice  (boiled)                72.5               2.8              o.i 

Shredded  Wheat           8.1             lo.s               1.4 

II. 5 
24.4 
77.9' 

0.7 
0.2 
2.1 

280 
498 
1660 

Graham                           s . 4 

Crackers                  Oatmeal                          6.3 

Soda                                 S-9 

10. 0              9.4 

II. 8             II. I 

9.8               9.1 

73.81 
69.  oi 

73.1' 

1-4 
1.8 
2. 1 

1904 
1920 
187s 

ri„-^                         White                              35.3 
^^^^^                        Graham                          35. 7 

9.2                1.3 
8.9               1.8 

53. 1' 
52. i> 

I.I 
I.S 

1182 
1189 

Boiled                       !     75.5 

T>  t  t„„^                   Mashed                         75.1 
Potatoes                   (,j^.p3                        1       22 

Sweet                              S  r  .  9 

2.5  i        0.1 

2.6  3.0 
6.8             39.8 
3.0        1        2.1 

20.9' 
17.8 
46.7 
42.1 

1.0 
1.5 
4.5 
0.9 

429 

493 

2598 

903 

White 
Eggs  (hen)               g°^jj.g  g^ijjig  pQ^. 
tion. 

86.2 
49.5 

73.7 

12.3  0.2 
15.7             33.3 

13.4  10.5 

0.6 
I.I 

1.0 

231 
1643 

672 

Milk 

87.0 

3.3               4.0 

so 

0.7               314 

Butter 

12.7 

1.3 

84.0 

1.92 

3450 

1  Peanut  butter 

2. 1 

29-3 

46. 5 

17. 1 

S.o 

2741 

Consomm6 

0^ ^„                          Tomato 

Soups                         pga 

j  Celery  (cream) 

96.0 
90.0 
86.9 
88.6 

■ 

2.5 
1.8 
3.6 
2.1 

I.I 
0.7 
2.8 

7.6 
5.0 

I.I 
1.5 
1 .2 
1.5 

S3 
179 
232 
243 

Tapioca  pudding 

64. 5     j          3.3 

3.2 

28.2 

0.8 

702 

Doughnuts                                                     18.3     ]         6.7 

21.0 

53.1^ 

0.9 

1942 

Ginger  snaps                                                   6.3              6.5 

8.6           76. oi 

2.6 

1848 

Peas  (cooked) 

73.8               6.7 

3.4     i      14.6 

I.S 

525 

Lettuce 

94-7     '          1.2 

0.3     I        2.91 

0.9 

87 

Apples                                                         84.6              0.4       1       0.5          14.2" 

0.3 

28S 

Oranges 

86 . 9               0.8 

0.2           II. 6 

o.S 

233 

Bananas 

75. 3                1.3 

0.6       1        22.0* 

0.8 

447 

Figs 

79. 1                i.S          18.8 

0.6 

368 

Sandwiches                Chfcken 

41.4  9.6        1      12.7      1      34. 5 

48. 5  12.3        '        5-4           32.1 

1.8 
1.7 

1319 
1026 

Cheese  (American) 

30.0             28.8        j     35.9             0.3 

5.08 

1990 

A  less  accurate  experiment  than  the  above  but  one  which  yields 
interesting  data  may  be  carried  out  as  follows: 

Ingest  a  simple  diet  whose  nitrogen  content  can  be  estimated  with  some 
degree  of  accuracy  (see  table  above).  Collect  the  urine  in  two-hour  periods 
from  7  A.  M.  to  ii  P.  M.  and  in  an  eight -hour  period  from   ii  P.  M.  to 

^Percentage  of  fiber,  included  under  carbohydrate:  shredded  wheat  (1.7),  Graham 
crackers  (1.5),  oatmeal  crackers  (1.9),  soda  crackers  (0.3),  white  bread  (0.5),  Graham 
bread  (i.i),  boiled  potatoes  (0.6),  doughnuts  (0.7),  ginger  snaps  (0.7),  lettuce  (0.7), 
apples  (1.2),  bananas  (1.0). 

2  Including  salt. 

^  Including  salt  and  sugar. 


METABOLISM  571 

7  A.  M.  and  analyze  for  total  nitrogen  or  urea.  The  next  day  ingest  the  same  diet 
plus  150-250  grams  of  lean  meat  whose  nitrogen  content  has  been  determined  by 
analysis  or  estimated.  Collect  the  urine  as  upon  the  previous  day  and  determine 
its  total  nitrogen  or  urea  content.  Plot  curves  showing  the  course  of  the  nitrogen 
or  urea  excretion  on  each  of  the  days.  How  soon  after  the  ingestion  of  the  large 
quantity  of  meat  did  you  note  an  increase  in  the  nitrogen  or  urea  excretion? 
How  many  hours  after  the  meal  was  the  maximum  quantity  of  nitrogen  or  urea 
excreted? 

8.  Influence  of  Purine-free  and  High  Purine  Diets. — The  uric  acid 
of  the  body  has  a  two-fold  origin,  i.e.,  it  may  arise  from  the  metabolism 
of  the  purine  (nuclein)  material  of  body  tissue  (glandular  organs  in 
particular)  or  it  may  arise  from  the  ingestion  of  purine  (nuclein) 
material.  That  uric  acid  which  arises  from  the  first  source  is  called 
endogenous  while  that  which  arises  from  the  second  source  is  termed 
exogenous.  Secretory  activity  may  also  act  to  increase  the  endogenous 
uric  acid  output.  The  urine  will  therefore  contain  uric  acid  even  though 
no  precursor  of  the  acid  be  ingested.  We  may  also  increase  the  uric  acid 
output  markedly  by  ingesting  a  high  purine  diet.  However,  no  matter 
how  much  purine  material  is  eaten  only  a  small  part  of  this  purine 
material  reappears  in  the  urine  as  uric  acid.  In  gout  it  is  claimed 
there  is  an  accumulation  of  uric  acid  in  the  blood  due  to  the  fact  that 
the  kidney  has  lost  the  ability  to  maintain  the  normal  blood  uric  acid 
level.  In  this  disease  the  excretion  of  uric  acid  may  be  low  before  an 
attack  and  increase  considerably  during  an  attack.  The  excretion  of 
exogenous  uric  acid  in  gout  is  also  much  slower  than  normal. 

Experiment. — Ingest  a  purine-free  diet  containing  about  16  grams  of  nitrogen 
and  consisting  of  egg,  cheese,  milk,  starch,  fruit,  sugar  and  water  for  a  period 
of  two  days  (for  purine  content  of  foods,  see  table,  page  572).  Determine  or 
estimate  the  nitrogen  content  (see  table,  page  569)  and  during  the  next  two 
days  substitute  sweetbreads,  thymus  or  Uver  for  all  the  nitrogen  of  the  diet 
maintaining  the  calorific  value  of  the  diet  the  same  as  before.  Return  to  the 
original  purine-free  diet  for  a  third  interval  of  two  days.  During  the  final  period 
of  two  days  feed  a  diet  of  sweetbreads  or  liver  containing  50  per  cent  more 
nitrogen  than  that  of  the  first  sweetbread  period.  Collect  the  urine  for  each 
of  the  eight  days  of  the  experiment  and  determine  uric  acid,  and  total  nitrogen 
or  urea.  Note  the  rise  in  the  uric  acid  output  during  the  sweetbread  periods. 
The  uric  acid  output  on  the  purine-free  diet  is  endogenous  in  origin.  Tabulate 
your  results.  The  following  data  were  secured  by  Taylor  and  Rose '  in  a  similar 
but  much  more  carefully  controlled  test  than  that  just  outlined. 

'  Taylor  and  Rose:  Jour.  Biol.  C/ictn.,  14,  419,  1913. 


572 


PHYSIOLOGICAL   CHEMISTRY 


INFLUENCE  OF  PURINE-FREE  AND  HIGH  PURINE  DIETS 

(Daily  Output) 


Urinary  constituents  determined 
(grams) 

Purine-free 
diet 

Purine  diet 
(medium) 

Purine  diet 
(increased) 

Purine-free 
diet 

Uric  acid 

0.09 

0. 14 

0.  24 

0.07 

Total  nitrogen 

8.9 

8.7 

9.1 

8.8 

UreaN  (+NH3) 

7-3 

7-1 

7-1 

7-oS 

Creatinine 

1-57 

1.49 

1-51 

PURINE  CONTENT  OF  FOODSi 
(Percentage  purine  base  nitrogen) 


Food 

Analyzed  by 

Food 

Analyzed  by 

Vogel* 

HalP 

Bessau 

and 
Schmid^ 

Vogel*     Hall' 

Bessau 

and 
Schmid* 

Beef    

O.O^O         O.OC2 

0.037 

Lettuce 

0.003 

Liver 

0.000 

0.110 

0.093 

Cucumbers 

None. 

Mutton 

0.026 

Rye  bread 

0.014   ! 

Trace. 

Tongue 

1 

0-0S5 

White  bread.... 

0.008 

None. 

None. 

Chicken 



0.029 

Milk 

0.0002 

0.C002 

None. 

Thymus 

0.398    0.403 

0.330 

Eggs 

None. 

None. 

None. 

Cod  fish 

0.040  '  0.023 

0.038 

Cheese 

0.0004 

None. 

Potatoes 

1 

0 .  001      0 . 0007 

0.009 

Rice 

0.0004 

None. 

None. 

'Celery 

1 



0.005 

Tapioca 

None. 

Peas 

o.oi6  i  0.016 

1 

0.018 

Oatmeal 

None. 

Spinach 

0.022    

0.024 

Onions 

None. 

Hominy 

0.004  1 

Tomatoes 

None. 

None. 

1  Other  purine-free  foods  not  listed  here  are  fruits,  butter,  cream  and  starch. 

^Vogel:  Miinch.  med.  Woch.,  58,  2433,  1911. 

3  Hall:  "The  Purine  Bodies,"  Philadelphia,  1904. 

*  Bessau  and  Schmid:  Therap.  Monatsch.,  March,  1910. 


METABOLISM 


573 


9.  A  Study  of  Endogenous  Uric  Acid  Output. — The  uric  acid  in  the 
urine  is  said  to  have  two  sources,  i.e.,  from  the  purine  material  of  the 
tissues  and  from  the  purine  material  ingested.  The  former  is  endogenous 
uric  acid,  the  latter  exogenous  uric  acid.^  The  output  of  uric  acid 
on  the  purine-free  diet  in  Experiments  571   and  574  is  endogenous. 


30 

"" 

20 

H 

10 

H 

^ 

A 

^  8  9  10  li  le 

2  3  4  ^  G  7  8  9 

Fig.  170 — IxFLCExcE  of  Protein  Ingestiox  on  Exdogenoxjs  Uric  Acid  Output. 
Gluten  (130  Grams)  ingested  at  i  P.M.  {Mendel  6*  Stehle:  Jour.  Biol.  Chem.,  22,  215, 
1915-) 

Mares"  claims  that  food-stuffs  act  to  increase  the  endogenous  uric 
acid  output  by  stimulating  the  digestive  glands  to  actiWty.  A 
similar  finding  is  reported  by  Mendel  and  Stehle.^  The  food-stuff 
having  the  most  pronounced  influence  was  protein.  Pilocarpine 
which  stimulates  the  digestive  glands  was  found  to  increase  the  endog- 
enous uric  acid  output  whereas  atropine  which  inhibits  secretory 
activity  was  found  to  decrease  the  output  of  endogenous  uric  acid. 


1 10 


^  °7  8  9  lOII  it  12  3  4  f  6  7  8  9   (H0UR5) 

.   Fig.  171 — The  Endogenous  Uric  .Acid  Output  during  Fasting.     {Mendel  is"  Stehle: 
Jour.  Biol.  Chetn.,  22,  215,  1915.) 

The  influence  of  protein  upon  the  endogenous  uric  acid  e.xcretion  is 
shown  by  the  chart  in  Fig.  170.  The  fasting  output  by  the  same 
individual  is  shown,  for  comparison,  in  Fig.  171. 

Experiment. — Ingest  a  purine-free  diet  consisting  of  millt,  egg,  fruit,  cheese, 
butter,  sugar  and  bread  for  one  day.  Continue  the  diet  for  breakfast  and  lunch- 
eon the  next  day  but  eat  nothing  after  12  o'clock  noon,  until  12  o'clock  noon  the 

*  Burian  and  Schur:  Zeit.  physiol.  Client.,  43,  532,  1904-5. 

*  Mares:  Arch.  f.  d.  ges.  Physiol.,  134,  59,  1910. 
*Mendl  andd  Stehle:  Jour.  Biol.  Chem.,  22,  215,  1915. 


574  PHYSIOLOGICAL   CHEMISTRY 

following  day,  i.e.,  the  third  day  of  the  experiment.  At  that  time  ingest  125-150 
grams  of  gluten  or  some  other  purine -free  protein  preparation.  On  the  fourth 
day  of  the  experiment  eat  nothing  imtil  9  P.  M. 

Collect  the  urine  each  day  in  hour  periods  from  7  A.  M.  to  9  P.  M.  and  analyze 
for  uric  acid  (see  methods  in  Chapter  XXVI).  Chart  your  data  similarly  to 
those  shown  in  Figs.  170  and  171,  page  573,  and  compare  them  with  the  findings 
there  recorded. 

10.  The  Rate  of  FHirine  Excretion.— The  purine  material  ingested 
by  the  average  normal  person  and  which  is  not  transformed  in  the  body 
will  be  eliminated  in  about  24  hours.  In  the  case  of  persons  afflicted 
with  gout  the  purine  elimination  is  delayed.  The  establishment  of  this 
delayed  purine  elimination  is  often  of  diagnostic  assistance. 

Demonstrate  the  rate  of  purine  excretion  as  follows:  Ingest  a  purine-free 
diet  consisting  of  egg,  milk,  cheese,  starch,  sugar,  fruit  and  water  for  two  days  and 
follow  this  by  a  day  in  which  sweetbreads,  thymus  or  Uver  is  substituted  for  one 
of  the  meals  of  the  day  (see  table  page  572  for  purine  content  of  foods).  Finish 
the  experiment  by  ingesting  the  original  purine-free  diet  for  two  days.  Collect' 
each  day's  urine  and  analyze  for  uric  acid.  How  soon  after  the  sweetbread 
ingestion  was  the  original  plane  of  endogenous  uric  acid  elimination  reestab- 
lished? If  one  desires  to  locate  this  time  more  definitely  the  urine  may  be 
collected  in  short  periods  (one  to  two  hours)  and  the  uric  acid  content  of  each 
specimen  determined.  Particularly  instructive  data  may  be  collected  by  per- 
forming the  above  experiment  on  a  gout  patient  and  upon  a  normal  person  for 
comparison.  • 

11.  A  Study  of  Creatinine  Elimination.— It  has  been  estabHshed 
that  a  normal  person  ingesting  a  creatinine-free  diet  will  excrete  a  uni- 
form quantity  of  creatinine  from  day  to  day.  The  daily  excretion  of  an 
adult  man  of  average  weight  ranges  from  1-1.5  grams.  For  data  as  to 
creatinine  excretion  of  a  60  kg.  man  see  Taylor  and  Rose's  figures  in 
table  on  page  572.  The  creatinine  excretion  depends  primarily  on  the 
active  mass  of  protoplasmic  tissue,  and  therefore,  it  is  generally  true 
that  a  fat  man  will  show  a  lower  creatinine  output  than  a  lean  man  of 
like  body  weight.     For  further  discussion  of  creatinine  see  Chapter  XXTL 

Experiment. — Ingest  an  ordinary  mixed  diet  (non-meat)  for  a  period  of  three 
days  varying  the  character  of  the  diet  daily.  Collect  the  urine  and  analyze  for 
creatinine.     (See  Chapter  XXVI  for  methods  of  analysis.) 

Did  the  creatinine  elimination  change  with  the  change  in  diet? 

12.  Influence  of  Water. — It  has  been  demonstrated  that  increased 
water  ingestion  influences  many  of  the  functions  and  activities  of  the 
human  body.^  The  increase  in  protein  catabolism  which  accomp- 
anies high-water  intake  is  shown  in  the  following  data  collected  from 

^  Hawk:  The  relationship  of  water  to  certain  life  processes  and  more  especially  to  nu- 
trition. Read  before  American  Philosophical  Society,  Philadelphia,  Feb.,  1914.  (See 
Bioch.  Bull.,  3,  420,  1914). 


METABOLISM 


:>/;> 


an  experiment  upon  a  normal  man.^     In  this  experiment  the  water  in- 
gestion at  meals  was  increased  3  liters  per  day  during  the  Water  Period. 

INFLUENCE  OF  HIGH- WATER  INTAKE  UPON  URINE  VOLUME  AND 
NITROGEN  PARTITION 


Day  of 
experi- 
ment 

Urine 
volume 

Nitrogen 

Urea-          Ammonia-      Creatinine-       Creatine- 
nitrogen         nitrogen         nitrogen          nitrogen 

Preliminary  Period 

4 
S 
6 

c.c. 
830 
920 
S80 

grams 
12.987 

12.084 
13-183 

grams 
"•338 
11.476 
11.568 

grams  grams  grams  ; 
0. 288              0.629           

0.305              0.619          

0.  ■?6o             cdiji          

Water  Period 

7 
8 

9 
10 
II 

3440 
3S40 
3670 
3610 
4020 

14.161 

13-491 
12.981 
12.976 
13-138 

12.596 

11-583 
II  .212 

11-455 
11.879 

0.486             0.610               0.063      i 
0.499             0.616               0.024      1 
0.553             0.589               0.102      1 
0.485             0.608               0.055 
0.456             0.589               0.128 

The  above  data  indicate  an  increased  catabolism  of  protein  material 
as  is  shown  by  an  increased  output  of  total  nitrogen  upon  the  first  and 
second  days  (days  7  and  8)  of  the  Water  Period.  Part  of  this  increase 
may,  however,  have  been  due  to  a  "flushing"  of  the  tissues  rather  than 
to  increased  catabolism  of  protein  structures. 

Experiments — (a)  Relation  of  Water  Intake  to  Volume  and  Specific  Gravity  of 
the  Urine. — Ingest  an  ordinary  mixed  diet  for  two  days.  Collect  the  urine  in  24- 
hour  periods.  During  the  first  day  ingest  very  little  fluid  of  any  kind  either  at 
meals  or  between  meals.  On  the  second  day  ingest  as  much  water  as  you  can 
without  physical  inconvenience.  A  person  of  average  size  should  have  no  difii- 
culty  in  drinking  5-6  quarts  per  day. 

Measure  the  volimie  of  each  day's  urine  and  take  the  specific  gravity.  Note 
the  pronounced  increase  in  volvune  and  the  low  specific  gravity  of  the  urine 
under  the  influence  of  high-water  ingestion. 

(b)  Influence  on  Protein  Catabohsm.  That  water  stimulates  protein  catabolism 
may  easily  be  demonstrated  as  follows:  Ingest  a  imiform  diet  imilk,  crackers, 
butter,  peanut  butter  and  water)  for  a  period  of  four  days.  During  the  fii  st  two  days 
ingest  your  customary  volume  of  water  per  day.  During  the  last  two  days  increase 
the  water  ingestion  to  5-6  liters  per  day.  Collect  urine  in  24-hour  periods  and 
analyze  for  total  nitrogen  by  Kjeldahl  method  (see  Chapter  XXVI  and  Note  on 
page  589).  Note  the  increased  excretion  of  nitrogen  under  the  influece  of  high- 
water  intake.  If  time  permits  other  nitrogenous  urinary  constituents  may  be 
determined  (see  table  above). 

1  Fowler  and  Hawk:  Jour.  Expt.  Med.,  12,  388,  1910. 


576'  PHYSIOLOGICAL    CHEMISTRY 

13  "Salt -free"  Diet. — In  order  to  be  properly  nourished  we  must 
ingest  a  certain  amount  of  inorganic  matter  daily.  If  we  fail  to  do 
this  our  metabolic  processes  become  abnormal  and  the  urine  is  one 
index  of  this  abnormah'ty.^ 

Experiment. — Ingest  an  ordinary  mixed  diet  containing  an  ample  salt  content 
for  a  period  of  two  days.  Follow  this  period  by  the  ingestion  of  a  diet  which  has 
had  its  salt  content  reduced  to  a  very  low  value.-  Sugar  and  ohve  oil  or  non- 
salted  butter  may  supply  the  bulk  of  the  calorific  part  of  the  diet  and  dialyzed  egg 
white  or  casein  or  commercial  protein  preparations,  e.g.,  plasmon,  gluten  or  gUdine 
may  supply  the  protein.  Ingest  such  a  diet  for  three  days.  (This  is  an  "acid- 
forming"  diet,  see  page  580.)  Collect  the  urine  and  analyze  for  sodium  chloride, 
acidity,  ammonia  and  total  nitrogen.  Compare  the  data  from  the  normal  days 
with  those  obtained  when  the  "salt-free"  diet  was  ingested.  Test  the  urine 
(Chapter  XXVIj  and  blood  (Chapter  XVI)  for  acetone.  An  acidosis  follows 
the  ingestion  of  a  salt-free  diet  for  a  sufficient  length  of  time. 

Did  you  feel  perfectly  normal  during  the  interval  you  were  ingesting  the 
"salt-free"  diet? 

14.  Salt -rich  Diet.- — On  an  ordinary  mixed  diet  a  normal  adult 
will  daily  excrete  10-15  grams  of  chloride,  expressed  as  sodium  chloride, 
in  the  urine.  On  a  salt-free  diet  this  excretion  decreases,  whereas  if 
the  diet  contains  an  excessive  quantity  of  sodium  chloride  this  excess 
will  be  promptly  excreted  in  the  urine.  Normal  feces  contain  very 
little  sodium  chloride  even  after  excessive  sodium  chloride  ingestion 
(see  Experiment  33). 

Experiment. — Ingest  an  ordinary  mixed  diet  for  two  days.  On  each  of  the 
following  two  days  take  a  similar  diet  plus  a  weighed  amoimt  (e.g.,  10  grams)  of 
sodium  chloride.  Collect  the  urine  for  the  four  days  in  24-hour  samples,  pre- 
serve and  analyze  for  sodium  chloride  (for  methods  see  Chapter  XXVI).  What 
proportion  of  the  added  chloride  was  recovered? 

If  it  is  desired  to  make  the  experiment  quantitative  in  character  ingest  a  uni- 
form diet  (see  Experiment,  page  592)  each  day  instead  of  the  ordinary  mixed  diet, 
and  examine  urine  and  feces  (see  Experiment  33)  for  chloride. 

15.  Acidosis. — Acidosis  may  be  induced  in  a  normal  person  by  the 
ingestion  of  a  "salt-free"  diet  such  as  described  in  Experiment  13, 
above,  or  by  the  ingestion  of  a  carbohydrate-free  diet.  The  acidosis 
appears  somewhat  earlier  under  the  latter  conditions.  The  non- 
carbohydrate  diet  is  rather  better  suited  for  the  demonstration  of 
acidosis  because  of  its  greater  palatability.  When  carbohydrates  are 
ingested  there  is  an  oxidation  of  fatty  acids  to  carbon  dioxide  and 
water.  When  no  carbohydrates  are  ingested  a  portion  of  the  fatty 
acids  are  converted  into  acetone  bodies.  These  are  difficult  to  oxidize 
and  are  excreted  as  such.     The  ketomiria  (excretion  of  acetone  and 

^  Taylor:  University  of  California  Publications,  Pathology,  i. 

'^  It  is  practically  impossible  to  secure  an  absolutely  "salt-free"  diet. 


MET.\BOLISiI  577 

diacetic  acid)  is  particularly  pronounced.  The  following  table  shows 
the  data  obtained  in  an  actual  case  of  the  withdrawal  of  carbohydrate 
food  from  the  diet  of  a  normal  man  (von  Noorden). 

ACIDOSIS  ACCOMPANYING  CARBOHYDRATE  WITHDRAWAL 


Day 

Diet 

Excretion  of  acetone  bodies  cal- 
culated as  ^-hydroxj-butyric 
acid  (grams) 

I 

Protein,  fat  and  carbohydrate. 

None. 

2 

Protein  and  fat. 

0.8 

1 

3 

Protein  and  fat. 

1. 9 

4 

Protein  and  fat. 

8.7 

5 

Protein  and  fat. 

20.0                                         I 

6 

Protein,  fat  and  carbohydrate. 

2.2 

Experiment. — Ingest  an  ordinary  mixed  diet  for  one  day.  Follow  this  by  a 
period  of  two  to  four  days  in  which  no  digestible  carbohydrate  is  eaten.  CA  diet 
of  meat,  eggs,  butter,  agar-agar  and  water  has  a  very  low  digestible  carbohydrate 
value.)  Collect  the  urine  for  each  day  of  the  experiment,  examine  it  quaUtatively 
for  acetone  bodies  (see  tests  in  Chapter  XXlll).  If  present,  determine  the  total 
acetone  bodies  quantitatively  (for  methods  see  Chapter  XXVI).  The  blood  may 
also  be  examined  (see  Chapter  XVI).  Did  the  withdrawal  of  carbohydrate  food 
cause  an  acidosis  or  ketonuria?  How  did  it  compare  with  the  acidosis  in  the 
above  table? 

i6.  "Alkaline  Tide." — For  a  time  after  a  meal  the  normal  acid 
reaction  of  the  urine  may  be  changed  to  neutral  or  alkaline.  This 
has  been  explained  as  due  to  the  withdrawal  of  hydrogen  ions  to  manu- 
facture the  hydrochloric  acid  of  the  gastric  juice. 

Experiment. — Ingest  an  ordinary  mixed  diet.  Urinate  just  before  dinner  and 
note  the  reaction  of  the  urine  to  htmus.  If  acid,  determine  the  hydrogen  ion 
concentration  by  the  method  given  in  Chapter  XXVI.  {li  alkaline,  discard  the 
urine  and  make  the  test  on  another  day.)  After  eating  a  heavy  dinner  (meats) 
collect  the  urine  at  intervals  of  a  half -hour  and  take  the  reaction  to  Utmus  and 
determine  the  hydrogen  ion  concentration  as  before.  Did  your  urine  change  in 
reaction  after  the  meal  and  if  so  how  long  a  period  elapsed  between  the  meal  and 
the  occurrence  of  the  maximimi  change  in  reaction? 

17.  The  "Partition"  of  Urinary  Nitrogen  and  Sulphur  as  Influenced 

by  Diet. — It  was  first  shown  by  Folin^  that  the  percentage  of  the  total 

nitrogen  and  total  sulphur  of  the  urine  which  appeared  in  the  form  of 

any  particular  nitrogenous  constituent  or  in  any  particular  form  of 

'  Folin:  Amer.  Jour.  Physiol.,  13,  118,  1905. 

37 


578 


PHYSIOLOGICAL    CHEMISTRY 


sulphur  was  regulated  directly  by  the  extent  of  the  total  nitrogen  and 
sulphur  elimination.  This  point  is  well  illustrated  in  the  following 
table  which  contains  data  regarding  the  so-called  "partition"  or 
"distribution"  of  the  urinary  nitrogen  and  sulphur. 

THE  NITROGEN  AND  SULPHUR  "  PARTITIONS"  AS  INFLUENCED  BY  DIET^ 


Constituent  of  the  urine 

Normal  protein  diet 

Starct 

i-cream 

diet 

Weight,  grams 

Nitrogen, 
grams 

Per  cent  of 
total  N  or  S 

Weight,  grams 

Nitrogen, 
grams 

CO 

M 

«-.      ° 

S3  *^ 
61.7 

Urea 

31.6 

14-7       87.5 

4.72 

2.  2 

1 
Ammonia 

0.6 

0.49:     3.0 

1 

1  0.51 

1 

0.42 

II-3 

Creatinine 

1-55 

0.58      3.6 

1. 61 

0.60 

17.2 

Uric  acid 

0.54 

0. 18       I.I 

0.27 

0.09 

2.5 

Undetermined 

0.8s       4.9 

0.27 

7-5  ' 

Total  N 

16.8 

100. 0 

3.6 

100. 0 

Inorganic  SO3 

3  27 

90.0 

0.46 

60.5 

Ethereal'sOa 

0.19 

5-2 

4-8 

0.  ID 

13-2 

1 

Neutral  SO3 

0.18 

0.  20 

26.3 

1 

Total  SO3 

3-^4 

100. 0 

0.  76 

100. 0  • 

It  will  be  observed  from  an  examination  of  this  table  that  a  normal 
protein  diet  which  gave  16.8  grams  of  urinary  nitrogen  yielded  87.5 
per  cent  of  this  nitrogen  as  urea,  3  per  cent  as  ammonia,  3.6  per  cent  as 
creatinine  and  i.i  per  cent  as  uric  acid;  whereas  a  "non-protein  diet" 
(starch  and  cream  containing  about  i  gram  of  nitrogen)  which  gave 
only  3.6  grams  of  urinary  nitrogen  yielded  only  61.7  per  cent  of  this 
nitrogen  as  urea  but  gave  a  greatly  increased  percentage  output  in  the 
case  of  each  of  the  other  nitrogenous  constituents  mentioned,  e.g.,  11. 3 
per  cent  as  ammonia,  17.2  per  cent  as  creatinine  and  2.5  per  cent  as 
uric  acid.  The  percentage  output  of  neutral  sulphur  was  also  greatly 
increased. 

It  will  furthermore  be  observed  that  the  actual  daily  output  of 
'  Folin:  Am.  Journ.  Physiol.,  13,  118,  1905. 


METABOLISM  579 

certain  of  the  constituents  is  uninfluenced  by  the  amount  of  protein 
ingested.  Among  these  are  creatinine  and  neutral  sulphur.  On  the 
other  hand  the  output  of  inorganic  sulphur  and  urea  is  more  or  less 
directly  proportional  to  the  protein  ingestion.  The  observation 
of  such  facts  as  these  led  Folin  to  formulate  his  theory  of  protein 
metabolism.^ 

Experiment. — During  a  period  of  two  or  three  days  ingest  an  ordinary  mixed 
diet  containing  100-125  grams  of  protein  (16-20  grams  of  nitrogen)  per  day. 
Collect  the  urine  accurately  in  24-hour  periods  (page  565)  preserve  it  and  analyze 
the  xirine  of  the  second  and  third  days  for  total  nitrogen,  urea,  creatinine,  total 
sulphur,  inorganic  sulphates,  ethereal  sulphates  and  neutral  sulphur  (by  differ- 
ence). For  methods  of  analysis  see  Chapter  XXVI.  Follow  this  period  by 
one  of  three  days  in  which  a  diet  of  starch  and  cream  having  a  similar  calorific 
value  is  ingested.  Analyze  the  urine  for  the  second  and  third  days  as  indicated 
above.  Calculate  your  results  and  tabulate  as  shown  in  the  table  on  page  578. 
How  did  the  change  in  the  diet  alter  the  metabolism  of  nitrogen  and  sulphur? 

In  calculating  the  calorific  value  of  a  diet  make  use  of  the  following  values : 

I  gram  protein 4.1  large  calories 

I  gram  fat 9.3  large  calories 

I  gram  carbohydrate 4.1  large  calories. 

18.  Protein-Sparing  Action  of  Carbohydrate  and  Fat. — The  non-nitrogenous 
nutrients,  carbohydrate  and  fat,  have  the  power  to  diminish  the  extent  of  the  catabo- 
lism  of  protein  in  the  normal  human  body.     In  other  words  they  are  said  to  ' '  spare 
protein.     This  point  is  illustrated  in  data  reported  by  von  Noorden  and  Dieters, 
which  are  tabulated  below. 

PROTEIN-SPARING  ACTION  OF  CARBOHYDRATE  AND  FAT 


Nitrogen  ingested 

Nitrogen  in  urine 

12.6  grams. 

10.4  grams. 

12.6  grams +200  grams  sucrose. 

9.0  grams.       13  per  cent  reduction  in  protein 
catabolism. 

It  will  be  observed  that  the  addition  of  200  grams  of  sucrose  to  the  diet  was 
accompanied  by  a  decrease  of  13  per  cent  in  the  amount  of  protein  catabolized. 
It  has  been  established  that  carbohydrates  are  more  efficient  "protein  sparers" 
than  are  the  fats.  For  example  Voit  found  carbohydrate  to  produce  a  9  per  cent 
decrease  in  protein  catabolism  whereas  fats  produced  only  a  7  per  cent  decrease. 

Experiment. — Ingest  a  uniform  diet  of  known  or  estimated  nitrogen  content 
for  a  period  of  four  days.  Collect  and  preserve  the  urine  accurately  (see  page  565) 
in  24-hour  samples  and  analyze  the  excretion  of  the  third  and  fourth  days  for  total 
nitrogen.     On  the  fifth  day  add  200  grams  of  sucrose  to  the  diet.     Analyze  this  urine 

^The  author's  article  on  "General  Considerations  of  Metabolism"  in  "Modern  Medi- 
cine" (Osier  and  McCrae)  2nd  Edition,  1914,  p.  594.  See  also  Folin:  American  Journal 
Physiol.,  13,  118,  1905. 


58o 


PHYSIOLOGICAL    CHEMISTRY 


also  for  total  nitrogen.     Calculate  your  results  and  tabulate  the  data  as  shown  in 
table  on  page  579. 

Did  the  sucrose  influence  the  catabolism  of  protein  in  your  body? 

19.  Hydrogen  Ion  Concentration  of  the  Urine  as  Influenced  by 
the  Ingestion  of  Acid-Forming  and  Base-Forming  Foods. — It  has  been 
demonstrated  by  Sherman  and  Gettler^  that  vegetables  and  fruits, 
on  burning,  leave  an  ash  in  which  the  basic  elements  (sodium,  potassium, 
calcium  and  magnesium)  predominate,  whereas  cereals,  meats  and  fish 
foods  leave  an  ash  in  which  the  acid-forming  elements  (chlorine,  sulphur 
and  phosphorus)  predominate.  A  list  of  acid-forming  and  base- 
forming  foods  is  given  in  the  following  table. 

EXCESS  OF  ACID-FORMING  OR  BASE-FORMING  ELEMENTS  IN  FOODS 

(Sherman  and  Gettler) 


Article  of  Food 


Excess  acid  or  base  in  terms  of 
normal  solutions.    Per  100  grams 


Acid  (c.c.) 


Base  (c.c.) 


Apples 

Asparagus 

Bananas 

Beans  (dried) 

Beans  (lima,  dried) 

Beets 

Cabbage 

Cantaloup 

Carrots. 

Cauliflower 

Celery 


Crackers 

Eggs 

Egg-white 

Egg-yolk 

Fish  (haddock). 
Lemons 


Lettuce 

Meat  (lean  beef) . 
Milk  (cow's) .... 

Oatmeal 

Oranges 

Potatoes 

Prunes 


Raisins 

Rice 

Wheat  (entire). 


7.81 

II. ID 

S-24 
26.69 
16.07 


13-91 


12.93 


8.10 
9.66 


3-76 

0.81 

5-56 

23.87 

41.65 

10.86 

4.34 

7-47 
10.82 

5-33 

7.78 


5-45 
7  37 

2-37 

S-6i 

7.19 

24-40* 

23.68 


1  Sherman  and  Gettler:  Jour.  Biol.  Cliem.,  11,  323,  191 2. 

"  Prunes,  plums  and  cranberries  yield  an  alkaline  ash  but  serve  to  htcrease  the  hydrogen 
ion  concentration  of  the  urine  because  of  their  benzoic  acid  content,  this  acid  being  syn- 
thesized with  glycocoU  in  the  kidney  and  elsewhere  to  form  hippuric  acid. 


METABOLISM  58 1 

The  above  data  indicate  that  potatoes,  oranges,  raisins,  apples, 
bananas  and  cantaloups  are  important  base-forming  foods.  Among 
the  most  important  acid-forming  foods  are  found  rice,  whole  wheat 
bread,  oatmeal,  meats  and  eggs.  Certain  fruits,  e.g.,  cranberries,^  prunes 
and  plums  yield  a  basic  ash  but  are  acid-forming  foods. 

This  is  due  to  the  fact  that  they  contain  benzoic  acid  which  is 
synthesized  with  glycocoll  in  the  body  to  produce  hippuric  acid  (see 
page  585).  It  is  worthy  of  note  that  some  plant  foods  are  base-formers 
and  others  are  acid-formers.  It  is  also  an  important  fact  that  acid 
fruits  yield  a  basic  ash  (see  page  580) . 

The  normal  diet  should  contain  sufhcient  base-forming  elements  to 
neutralize  the  acids  formed.  If  these  acids  are  not  neutralized  by 
the  basic  elements  in  the  diet  they  will  be  neutralized  by  the  fixed 
bases  of  the  tissues  of  the  body  and  a  seriously  deranged  metabolism 
may  result.  (See  experiment  on  "salt-free  diet,"  page  576.)  Organic 
salts  of  the  alkalis  (c.^.,  sodium  bicarbonate  or  sodium  acetate)  are  often 
given  therapeutically.  They  reduce  the  H  ion  concentration  of  the 
urine:  the  same  result  so  far  as  urine  reaction  is  concerned  may  be 
secured  by  feeding  properly  selected  base-forming  foods.  The  ingestion 
of  sodium  dihydrogen  phosphate  (NaH2P04)  will  increase  the  acidity 
of  the  urine:  a  like  result  may  be  produced  by  feeding  properly  selected 
acid-forming  foods.  Anything  which  produces  an  increase  in  the  H  ion 
concentration  of  the  urine  will  produce  an  increase  in  the  ammonia 
output. 

On  a  mixed  diet  the  H  ion  concentration  of  the  urine-  has  been 
found  to  be  6  ±  0.1.  ^  In  nephritis  the  H  ion  concentration  of  the 
urine  may  be  increased  to  5.33  or  higher.  Alkalis  have  been  used  with 
apparent  success  in  the  treatment  of  nephritis.^  It  is  evident  that  base- 
forming  foods  properly  selected  should  be  suitable  dietary  articles  for 
nephritics.^  For  a  detailed  discussion  of  acid-forming  and  base-forming 
foods  see  article  by  Blatherwick.^ 

Experiment, — Ingest  a  uniform  diet  consisting  of  milk,  crackers,  butter,  pea- 
nut butter,  and  water  in  desired  quantities  for  a  period  of  three  days.  Follow  this 
by  a  period  of  six  days  during  the  first  three  of  which  considerable  quantities  of 
acid-forming  foods  (see  table  page  580)  are  added  to  the  diet.     During  the  second 

^  Radin  reports  this  berry  to  contain  0.06  per  cent  benzoic  acid  (Blatherwick:  Arch. 
Int.  Med.,  14,  409,  1914). 

^Henderson  and  Palmer:  Jour.  Biol.  Clicm.,  14,  81,  1913. 

'  H  ion  concentration  may  be  expressed  as  gram  of  ionized  H  per  liter  of  water.  A  neu- 
tral solution  has  a  H  ion  concentration  of  iXio"',  or  0.000,000,1  gram  per  liter.  It  is 
often  customary  to  express  the  H  ion  concentration  according  to  Sorensen's  logarithmic 
notation.  For  example  instead  of  expressing  the  H  ion  concentration  of  a  neutral  solution 
as  I X  io~^  he  expresses  it  as  7.00.  An  increasing  H  ion  concentration  decreases  this  value 
and  an  increasing  OH  ion  concentration  increases  the  value. 

■•  Fisher:  Nephritis,  New  York,  191 2. 

*  Blatherwick:  Arch.  Inl.  Med.,  14,  409,  1914. 


582 


PHYSIOLOGICAL   CHEMISTRY 


half  of  the  period  (days  four  to  six)  add  an  abundance  of  base -forming  foods  to  the 
diet.  Distilled  water  should  be  used  for  drinking  purposes  and  a  uniform  volimie 
should  be  ingested  daily.  Collect  the  urine  in  24-hour  periods,  preserve  and 
analyze  for  H  ion  concentration,  titratable  acidity  and  ammonia  (for  methods 
see  Chapter  XXVI).  Compare  your  results  with  those  tabulated  in  the  table 
below. 

REACTION  OF  URINE  AS  INFLUENCED  BY  DIET^ 


Basal 
diets- 


4 


Determi- 
nation. 


Baked  pota- 

No.  I  No.  2    i,°^il?f>. 
grams  per 

day)+basal 

I  diet  No.  I 

,  S  S         (6  days) 

days  !  days 


Rice  (210       Cranberry  |     Bread' 
grams  per    sauce  (300-        (whole 
day)  +basal    600  grams    wheat)  450 
diet  No.  I     per     day)  +  grams  for  i 
(4  days)        basal  diet,    day  +  basal 
No.  I        diet.  No.  i 
;     (6  days)      | 


Prunes        Cantaloup* 
(330-550     (260     grams 
grams  per    per     day)  + 
day)  +basal    basal  diet, 
diet.  No.  2  No.  2 

(3  days)  (5  days) 


H  ion  con-  7  19 
centration. 

5.57 

7.74 

7. 4^-6. 90 

7.14 

'   6.30-5.70 
6.19 

!                 1                  i 

6.80      1  s. 30-4. 80  '  5.30-7.38 

(Previous        ■ ■        ■ ' 

day     6.90)         5.07                 6.70 

Titratable 

aciditv         275 
(c.c.    N/io 

474 

196-216 
203 

166-297 
233 

391-488 
407 

350         570-540-578      466-250 

(Previous      ■ ■     ■ • 

day  297)             563                   328 

Ammonia                           0 
N  (grams)  0.310  0.464  — 

221-0. 248 
.   .  - 
0.238 

0.166-0. 251 
0.198 

0.219-0. 391 
0.30s 

0.280        0.602-0.729  o.s 13-0. 220 

;day  0.251)         0.654              0.310 

20.  Hydrogen  Ion  Concentration  of  the  Urine  as  Influenced  by 
Alkali  and  Acid  Ingestion. — The  ingestion  of  certain  organic  salts  of  the 
alkaHs,  e.g.,  sodium  citrate  and  sodium  bicarbonate  will  cause  a  decrease 
in  the  hydrogen  ion  concentration  of  the  urine.     The  ingestion  of  acids 


INFLUENXE  OF  INGESTED  SODIUM  BICARBONATE  ON  H  ION 
CONCENTRATION  OF  URINE 


Experiment 
Number 

Sodium 
Bicarbonate, 

Hydrogen  Ion  Concen- 
tration before  Bicarbon- 

Time  of  Collection  of  Specimen, 
of  Urine  and  H  Ion  Concen- 
tration 

Grams                   ate  Ingestion 

11.00 
A.M. 

12.00 
noon 

1. 00 
P.M. 

2 . 00   3 . 00 
P.  M.P.  M. 

1 

I 
2 
3 
4 
S 
6 

4 
8 
12 
8 
8 

7.40 

5 -40 

5-30 
7.40 

5-85 
6.70 

8.30 

8.50 
8.70 
8.50 

7.48 
8.30 
8.70 
8.70 

7.48 
6.50 
8.70 
8.50 
8.70 
8.50 

7.40 
6.50 
8.70 
8.50 
8.70 
8.70 

5.85 

7-40 

8.70  ' 

8.50 

8.30 

8.50 

8 

7.48 

8.70 

1  Tabulated  from  data  reported  by  Blatherwick  (Arch.  Int.  Med.,  14,  409,  i9i4)' 
Experiments  all  made  on  the  same  subject  (B). 

^  Basal  diet  No.  i  contained  100  grams  Graham  crackers,  25  grams  butter,  400  c.c.  whole 
milk  ingested  at  each  of  the  three  dailj-  meals.  One  apple  and  one  soft  boiled  egg  added  at 
supper.     In  diet  No.  2  whole  wheat  crackers  were  substituted  for  the  Graham  crackers. 

*  This  day  was  preceded  by  NaHCOa  ingestion  for  three  days  and  by  rice  ingestion  for 
four  days. 

*  This  diet  followed  immediately  after  the  diet  of  prunes  (see  5). 


MET.ABOLISM 


583 


(either  organic  or  inorganic)  or  acid  salts,  e.g.,  sodium  dihydrogen  phos- 
phate will  increase  the  hydrogen  ion  concentration  of  the  urine.  The 
alkahs  are  much  more  effective  in  producing  changes  in  reaction  than 
are  the  acids.  The  influence  of  ingested  alkali  (sodium  bicarbonate)  is 
shown  in  the  foregoing  table  containing  data  submitted  by  Henderson 
and  Palmer.^ 

Blatherwick-  reports  a  decrease  in  ammonia  nitrogen  output  from 
0.256  gram  to  0.072  gram,  and  an  accompanying  decreased  acidity 
under  the  influence  of  bicarbonate  ingestion  (25  grams  in  two  days). 

The  influence  of  ingested  acid  (benzoic)  is  shown  in  the  following 
data  reported  by  Blatherwick.- 

INFLUENCE  OF  BENZOIC  ACID  INGESTION" 


Day 

Titratable  acidity 
(c.c.  N/io) 

H  ion  concentration 

Ammonia  N  (grams) 

1                                                 1 
I                            392                                         6.15 

0.292                ! 

2                            410 

6.15 

0.374 

3                                443 

6.00 

0.422               ' 

4       J                         434                                             6.00 

0.408 

5                                468                                              5.70 

0.418 

(For  further  discussion  of  dietary  alterations  of  urine  reaction  see 
preceding  experiment.) 

Experiments. — (a)  Influence  of  Alkali. — Ingest  a  uniform  diet  consisting  of 
milk,  crackers,  butter,  peanut  butter  and  distilled  water  for  a  period  of  two 
days.  During  the  next  two  days  take  the  same  diet  and  ingest  24  grams  of 
sodiimi  bicarbonate  between  meals  (12  in  A.  M.  and  12  in  P.  M.).  Collect  the 
urine  in  24-hour  periods  and  analyze  it  for  titratable  acidity,  H  ion  concentra- 
tion and  ammonia.  Compare  yovu:  results  with  those  shown  in  table  on 
page  582. 

If  desired  the  bicarbonate  may  be  given  in  one  dose  of  8-12  grams  and  the 
urine  collected  in  hourly  specimens  for  the  next  five  hours  and  each  specimen 
analyzed.    Data  from  such  experiments  are  shown  in  table  on  page  582 . 

(b)  Influence  of  Acid. — Proceed  as  above  except  that  i  gram  of  benzoic  acid 
(in  capsule)  is  ingested  before  each  meal  of  the  experimental  period. 

The  experiment  may  also  be  varied  by  ingesting  10  grams  of  sodium  dihydro- 
gen phosphate  early  in  the  day  and  collecting  the  urine  in  hoiurly  fractions  or  in 
one  24-hour  sample. 

^  Henderson  and  Palmer:  Jour.  Biol.  Cliem.,  14,  81,  1913. 
^See  p.  581. 

"  One  gram  of  benzoic  acid  in  a  capsule  before  each  meal.  Basal  diet  Xo.  i  described  on 
page  582  was  used. 


584  PHYSIOLOGICAL   CHEMISTRY 

From  yotir  experiments  what  do  you  conclude  as  to  the  relative  eflBciency  of 
acid  and  alkali  in  altering  the  reaction  of  the  urine? 

21.  Influence  of  a  High  Calorie  Non -Nitrogenous  Diet. — If  an 

individual  fasts  there  is  a  combustion  of  a  certain  amount  of  protein 
tissue  each  day  of  the  fast.  The  destruction  of  such  tissue  is  rather 
low  on  the  first  day  due  to  the  fact  that  the  glycogen  stores  of  the  body 
are  being  utilized  to  furnish  the  necessary  energy.  If  an  individual 
instead  of  fasting,  ingests  a  diet  of  high  calorific  value  and  very  low  in 
nitrogen  the  output  of  nitrogen  in  the  urine  of  the  third  or  fourth  day 
will  be  less  than  on  the  third  or  fourth  day  in  fasting.  This  is  due  to 
the  fact  that  the  body  derives  sufficient  energy  from  the  high  calorie 
diet  and  there  is  less  destruction  of  protein  body  tissues  than  occurs  in 
fasting.  For  a  discussion  of  energy  value  of  foods  see  "Determination 
of  Fuel  Value  of  Foods,"  below,  and  the  table  on  page  569. 

Experiment. — ^Ingest  a  high  calorie  diet  which  is  very  low  in  nitrogen  or 
actually  non-nitrogenous.  A  satisfactory  diet  may  include  sugar,  butter,  starch, 
cream,  agar-agar  and  water.  (For  energy  values  see  below  and  table,  page 
568.)  Ingest  such  a  diet  for  three  days.  Collect  the  urine  in  24-hour  periods, 
preserve  and  analyze  it  for  total  nitrogen,  acidity  and  ammonia.  Note  the  low 
nitrogen  excretion  on  the  third  day  as  compared  with  the  nitrogen  output  of  the 
third  day  of  fasting.  If  so  desired,  you  may  (at  some  later  date)  fast  for  three 
days  and  repeat  the  above  analyses  for  comparison. 

Determination  of  Fuel  Value  of  Food. — When  organic  substances  are  oxidized 
or  burned  in  the  human  body  they  liberate  a  certain  amount  of  heat.  This  calorific 
energy  or  heat  value  varies  according  to  the  type  of  organic  matter  undergoing 
oxidation.  Thus  the  proteins,  fats  and  carbohydrates  of  the  diet  when  they  are 
burned  in  the  body  yield  different  quantities  of  heat  per  unit  of  substance  than  do 
organic  acids,  alcohol,  etc.  The  energy  values  of  pure  protein  fat  and  carbohydrate 
are  the  following: 

Protein  =41  large  calories  per  gram. 

Fat  =9-3  large  calories  per  gram. 

Carbohydrate  =  4.1  large  calories  per  gram. 

In  arriving  at  the  energy  value  of  any  given  diet  it  is  customary  to  burn  weighed 
samples  of  the  various  foods  in  an  oxygen  atmosphere  in  an  apparatus  called  a 
bomb  calorimeter  (see  Fig.  172,  page  586).  By  this  means  we  may  determine  how 
much  heat  is  liberated  when  the  ingested  food  is  oxidized  in  the  body.  A  correction 
must  be  made  for  the  incompletely  oxidized  substances  of  the  urine  and  feces.  A 
large  mass  of  data  concerning  the  heat  value  of  foods  has  been  collected  and  tabu- 
lated, and  it  is  therefore  possible  to  arrive  at  an  approximate  idea  of  the  energy 
value  of  a  diet  by  calculation  (see  table,  page  569).  The  bomb  colorimeter  shown 
in  Fig.  172,  page  586,  is  one  of  the  most  satisfactory  for  actual  determination  of  the 
heat  of  combustion  of  organic  substances. 

22.  Metabolism  in  Fasting.— The  metabolism  of  a  fasting  man  is 
entirely  different  from   the  metabolism  of  a  well-nourished  person. 


METABOLISM 


O^O 


The  collection  and  analysis  of  the  urine  during  a  short  fast  (three 
to  seven  days)  will  demonstrate  many  important  facts.  The  following 
table,  which  contains  data  from  fasting  tests  made  in  the  author's 
laboratory/  illustrates  some  of  the  points  in  which  fasting  metabolism 
differs  from  normal  metabolism: 


METABOLISM  IN  FASTING 

Day 

Body 

Total 

Ammonia 

Creatine 

Acidity. 

P2O6 

Chloride 

of 

weight, 

X 

N 

X 

c.c.  N/io 

grams, 

period 

H. 

grams 

grams 

grams 

XaOH 

XaCl 

Preliminary  Feeding  Period 

1-4 

Av.  74.16 

10.430 

0.112 

None 

238.6 

2.768 

9.007 

Fasting  Period 

I 

73-32 

10.072 

0.288 

0.269 

328.9 

2.616 

5-035 

2 

71.98 

15-072 

0.642 

0.073 

677.1 

2.509 

3-231 

3 

70.92 

14-463 

0.862 

0.089 

770.4 

2.851 

2-539 

4 

70.24 

13.080 

1. 201 

0.068 

664.2 

2.490 

I -253 

5 

69.61 

I I. 801 

1.266 

0.033 

5250 

2.376 

1-474 

6 

69.12 

II. 214 

1-373 

0.022 

462 . 4 

1. 186 

1-132 

7 

68.70 

10-734 

1-371 

0.003 

438.9 

0955 

I-I37 

Abstinence  from  food  for  a  few  days  can  in  no  way  operate  to  the 
disadvantage  of  a  normal  person.  In  fact  indi\dduals  affected  with 
certain  types  of  gastro-intestinal  disorders  are  benefited  by  fasting. 
The  fasting  treatment^  is  also  being  used  with  success  in  cases  of  diabetes 
mellitus. 

In  order  to  determine  experimentally  how  the  fasting  metabolism  differs  from 
normal  metaboUsm  proceeds  as  follows :  Ingest  an  ordinary  mixed  diet  and  col- 
lect your  urine  (see  page  565)  for  a  day.  Measure  the  volimie  and  analyze  the 
sample  for  total  nitrogen,  ammonia,  creatine,  sodium  chloride,  total  phosphates 
and  acidity'  (for  methods  see  Chapter  XXVI).  For  the  next  few  days  (three  to 
seven  as  desired)  ingest  nothing  but  water  and  collect  the  urine  accurately  and 
analyze  for  the  constituents  enumerated  above.  Tabulate  your  results  and 
compare  them  with  those  given  in  the  table  above. 

23.  Synthesis  of  Hippuric  Acid  in  Human  Body. — Hippuric  Acid  is 
present  in  human  urine  in  small  amount,  about  0.7  gram  being  excreted 

'  The  chloride,  phosphate  and  acidity  determinations  were  collected  during  one  seven- 
day  fast  and  the  other  data  collected  during  a  different  fast  on  the  same  man.  (See 
Howe,  Mattill  and  Hawk:  Jour.  Amer.  Chem.  Soc,  3$,  568,  191 1;  and  Wilson  and  Hawk: 
Jour.  Amcr.  Chem.  Soc,  36,  137,  1914.) 

-Allen:  Amer.  Jour.  Med.  Set.,  150,  480,  1915. 

'  A  more  accurate  e.\periment  may  be  carried  out  by  ingesting  a  uniform  diet  of  known 
composition  (see  page  569)  for  a  few  days  before  the  fast. 


586 


PHYSIOLOGICAL   CHEMISTRY 


per  day.  The  urine  of  herbivorous  animals  contains  much  larger  quan- 
tities. This  acid  is  formed  in  the  animal  body,  by  synthesis  from  ben- 
zoic acid  and  glycocoll  which  takes  place  in  the  kidneys  and  elsewhere.^ 

Experiment. — Ingest  2  grams  of  sodium  benzoate  or  ammoniimi  benzoate 
before  retiring  at  night.  Collect  the  first  fraction  of  urine  voided  the  next  morn- 
ing.   The  benzoate  has  been  synthesized  with  glycocoU  to  form  hippuric  acid. 


Fig.    172. — -BERTHELOT-AxWATKli    BoMB    CALORIMETER. 

The  urine  will  therefore  be  found  to  contain  much  more  of  this  acid  than  is  nor- 
mally present.     Isolate  the  hippuric  acid  by  one  of  the  following  methods : 

(a)  First  Method. — Render  the  urine  alkaline  with  milk  of  lime,  boil  for  a  few 
moments  and  filter  while  hot.  Concentrate  the  filtrate,  over  a  burner,  to  a  small 
volume.  Cool  the  solution,  acidify  it  strongly  with  concentrated  hydrochloric  acid 
and  stand  it  in  a  cool  place  for  24  hours.  Filter  off  the  crystals  of  hippuric  acid 
which  have  formed  and  wash  them  with  a  little  cold  water.  Remove  the  crystals 
^Kingsbury  and  Bell:  Jour.  Biol.  Chem.,  21,  297,  1915. 


METABOLISM  587 

from  the  paper,  dissolve  them  in  a  very  small  amount  of  hot  water  and  percolate 
the  hot  solution  through  thoroughly  washed  animal  charcoal,  being  careful  to  wash 
out  the  last  portion  of  the  hippuric  acid  solution  with  hot  water.  Filter,  concen- 
trate the  filtrate  to  a  small  volume  and  stand  it  aside  for  crystallization.  Examine 
the  crystals  under  the  microscope  and  compare  them  with  those  in  Fig.  125,  page  389. 
This  method  is  not  as  satisfactory  as  Roaf's  method  (see  below). 

{b)  Roaf's  Method. — Place  the  urine  in  a  casserole  or  precipitating  jar  and  add 
an  equal  volume  of  a  saturated  solution  of  ammonium  sulphate  and  1.5  c.c.  of 
concentrated  sulphuric  acid  per  100  c.c.  of  urine.  Permit  the  mixture  to  stand  for 
twenty-four  hours  and  remove  the  crystals  of  hippuric  acid  by  filtration.  Purify 
the  crystals  by  recrystallization  according  to  the  directions  given  above  under  First 
Method.  Examine  the  crystals  under  the  microscope  and  compare  them  with  those 
given  in  Fig.  125,  page  389. 

It  is  possible,  by  the  above  technic,  to  isolate  hippuric  acid  in  crystalline  form 
from  as  small  a  volume  as  25-50  c.c.  of  herbivorous  urine.  The  greater  the  amount 
of  ammonium  sulphate  added  the  more  rapid  the  crystallization  until  at  the  satura- 
tion point  the  crystals  of  hippuric  acid  sometimes  form  in  about  ten  minutes. 

n.  METABOLISM  PROCEDURES  INVOLVING  THE 
MANIPULATION  OF  THE  FECES^ 

24.  "Separation"  of  Feces. — In  order  to  differentiate  the  feces 
which  correspond  to  the  food  ingested  during  any  given  interval  it  is 
customary  to  cause  the  person  under  observation  to  ingest  some  sub- 
stance, at  the  beginning  and  end  of  the  period  in  question,  which  shall 
sufficiently  differ  in  color  and  consistency  from  the  surrounding  feces  as 
to  render  such  differentiation  comparatively  easy.  Two  "markers" 
very  widely  used  in  such  tests  are  wood  charcoal  and  carmine.  In 
making  an  actual  separation  of  feces  in  a  metabolism  experiment 
proceed  as  follows:  Just  preceding  or  in  the  early  part  of  the  first  meal 
(usually  breakfast)  of  the  metabolism  test,  ingest  a  gelatine  capsule 
(No.  00)  containing  0.2-0.3  gram  of  carmine  or  charcoal.  From  this 
time  collect  all  stools  in  flat-bottom  porcelain  dishes  and  examine  for  the 
presence  of  the  "marker."  All  fecal  matter  containing  portions  of  the 
marker  may  be  considered  as  representing  the  diet  in  question.  This 
fecal  matter  should  be  retained  and  preserved  (see  page  588).  Just 
before  or  in  the  early  part  of  the  first  meal  (usually  breakfast)  following 
the  end  of  the  metabolism  test  a  second  "marker"  in  a  gelatine  capsule 
should  be  ingested.  The  feces  should  be  carefully  inspected  until  the 
marker  makes  its  appearance.  Retain  all  fecal  matter  uncolored  by 
the  marker,  reject  the  remainder.  Frequent  difficulties  are  encountered 
in  the  practical  separation  of  feces,  but  the  character  of  such  difficulties 
will  be  most  satisfactorily  impressed  by  the  performance  of  actual 
separations. 

'  For  other  practical  work  on  feces  see  Chapter  XIV. 


588  PHYSIOLOGICAL   CHEMISTRY 

25.  Collection  and  Preservation  of  Feces  and  the  Mixing  and 
"Weighing  for  Analysis. — The  older  methods  in  vogue  in  metabolism 
work  embraced  the  analysis  of  dried  feces.  Various  investigators  later 
demonstrated  that  the  drying  of  feces  was  accompanied  by  losses  and 
changes  of  some  of  the  organic  constituents' of  the  feces.  ^  Therefore 
the  chemical  examination  of  all  stools  wherever  possible  should  be 
made  on  the  fresh  feces.  If  a  study  is  being  made  which  extends  over 
several  days  and  it  is  desired  to  economize  time  and  effort  in  the 
chemical  examination  the  daily  fecal  output  or  an  aliquot  portion  of  each 
stool  may  be  collected  in  a  friction-top  can  or  pail  of  suitable  size  and 
preserved  by  thymol  and  refrigeration.^  This  method  has  been  found 
satisfactory  when  the  feces  are  to  be  examined  for  inorganic  constituents 
or  total  nitrogen.  For  the  determination  of  fat,  carbohydrate,  etc., 
the  fresh  stool  should  be  employed. 

In  the  preservation  of  feces  for  the  determination  of  total  nitrogen 
the  following  simple  procedure  may  be  used:  Introduce  each  stool  into 
a  weighed  friction-top  can  or  pail  and  place  the  vessel  in  a  cold  room  or 
refrigerator.^  At  the  end  of  the  period  mix  the  feces  thoroughly  and 
analyze  weighed  portions.  In  case  individual  stools  are  analyzed,  the 
stool  should  be  collected  in  a  weighed  flat-bottom  porcelain  dish."*  After 
mixing  the  feces  very  thoroughly  the  weight  of  dish,  spatula  and  feces  is 
determined  and  the  weight  of  the  feces  secured  by  difference.^  A  por- 
tion of  the  well-mixed  feces  is  then  introduced  into  a  large  weighing 
bottle  containing  a  glass  hoe.  Desired  amounts  of  feces  are  then 
removed  for  analysis  and  the  exact  weight  of  such  amounts  obtained  by 
difference. 

26.  Bacterial  Nitrogen  in  Feces.^ — About  50  per  cent,  of  the  total  nitrogen  of 
the  feces  is  made  up  of  bacterial  cells  (see  Chapter  XIV  on  Feces).  To  demon- 
strate this  point  proceed  as  follows: 

(a)  Ingest  an  ordinary  mixed  diet.  Collect  a  representative  stool  from  this 
diet  and  after  mixing  it  thoroughly  separate  the  bacterial  cells  from  a  weighed  por- 
tion as  described  in  Chapter  XIV.  After  examining  some  of  the  suspension  under 
the  microscope  and  noting  the  bacterial  cells  determine  the  bacterial  nitrogen  in 

^  Zaitschek:  Pflugers  Arch.,  98,  595,  1903. 
Schimidzu:  Bioch.  ZeiL,  28,  237,  1911. 
Konig:  Landw.  Vers.  Slat.,  38,  230. 

Frear  and  Holter:  Report,  Penn.  Stale  College,  p.  123,  1891. 
Emmett  and  Grindley:  Jour.  Am.  Cheni.  Soc,  31,  570,  1909. 
-  Howe,  Rutherford  and  Hawk:  Jour.  Amer.  Chem.  Soc,  32,  1683,  1910.     This  proce- 
dure is  not  satisfactory  if  fat  is  to  be  determined  (Smith,  Miller  and  Hawk:  Jour.  Biol. 
Chem.,  21,  395,  1915).     Such  feces  shows  an  hydrolysis  of  fat  to  fatty  acid  and  a  decrease 
in  total  fat. 

';  The  author  uses  a  brine  tank  at  —  i2°C.  in  which  the  feces  are  quickly  frozen. 
*  The  spatula  for  mixing  the  feces  should  be  weighed  with  the  dish. 
^In  case  it  is  desired  an  aliquot  part  of  each  stool  may  be  placed  in  a  friction-top  can 
or  pail  and  preserved  as  a  "composite  sample"  for  the  period. 


METABOLISM  589 

the  entire  volume  of  suspension  by  the  Kjeldahl  method^  (see  Chapter  XXV'I). 
Also  determine  the  total  nitrogen  in  weighed  portions  of  the  original  feces  by  the 
Kjeldahl  method.  What  percentage  of  the  total  nitrogen  of  the  feces  is  bacterial 
nitrogen? 

(b)  If  it  is  desired  to  determine  the  actual  amount  of  nitrogen  which  is  excreted 
daily  in  the  feces  in  the  form  of  bacterial  cells,  proceed  as  follows:  Ingest  an  ordi- 
nary mixed  diet  for  a  period  of  three  days.  Separate  the  feces  for  this  period  accord- 
ing to  directions  given  on  page  587,  using  charcoal  for  the  first  separation  and 
carmine  for  the  second  or  vice  versa.  Preserve  the  feces  for  the  period  according 
to  directions  given  on  page  588.  Mix  the  weighed  feces  thoroughly  and  analyze 
for  bacterial  nitrogen  and  total  nitrogen  according  to  directions  given  elsewhere- 
(see  Chapters  XIV  and  XXVI).  Calculate  the  actual  output  of  bacterial  nitrogen 
per  day  and  the  percentage  of  the  total  nitrogen  of  the  feces  which  was  excreted 
per  day  in  the  form  of  bacterial  nitrogen. 

27.  "Metabolic  Product"  Nitrogen  in  Feces. — A  certain  quota  of  the  fecal 
nitrogen  is  due  to  the  presence  of  residues  of  digestive  secretions,  epithelial  cells, 
bacteria,  etc.  The  nitrogen  in  these  forms  has  been  called  "metabolic  nitrogen." 
To  determine  this  form  of  nitrogen  proceed  as  foUows:  Ingest  a  non- nitrogenous 
diet  for  a  period  of  two  days.  The  diet  may  include  desired  quantities  of  starch, 
cream,  sugar,  butter,  water  and  sodium  chloride.  About  15  grams  of  agar-agar 
should  be  added  to  the  diet  to  prevent  constipation  and  to  insure  the  evacuation 
of  approximately  the  normal  quantity  of  feces.  (For  inJfluence  of  agar-agar  see 
Experiment  28.)  To  separate  the  feces  properly  ingest  a  capsule  of  carmine  at 
the  beginning  of  the  test  and  one  of  charcoal  at  the  end  (see  page  587).  Preserve 
the  feces  as  described  on  page  588.  After  mixing  the  feces  thoroughly  determine 
the  nitrogen  in  weighed  quantities  by  the  Kjeldahl  method^  according  to  direc- 
tions given  in  Chapter  XXVI.  Calculate  the  quantity  of  nitrogen  eliminated  per 
day.  Inasmuch  as  no  nitrogen  was  ingested  the  nitrogen  present  in  the  feces  is 
of  metabolic  origin,  i.e.,  it  is  made  up  principally  of  nitrogen  in  the  form  of  cells, 
digestive  secretions  and  bacteria. 

28.  Influence  of  Indigestible  Non-Nitrogenous  Material  upon 
Fecal  Output. — This  may  be  demonstrated  by  agar-agar  ingestion. 
This  indigestible  hemicellulose  has  the  property  of  absorbing  water 
readily  and  therefore  when  ingested  it  increases  the  bulk  of  the  feces 
considerably.  This  fact  is  made  use  of  in  some  forms  of  constipation 
and  in  the  determination  of  metabolic  product  nitrogen  (see  Experi- 
ment 27). 

Experiment. — Ingest  a  uniform  diet  for  four  days.  Divide  the  interval  into 
periods  of  two  days  each,^  and  "separate"  the  feces  by  charcoal  or  carmine  (see 
Experiment  24).  On  the  third  and  fourth  days  ingest  10  g^ams  of  agar-agar  at 
each  meal.  Collect  the  feces  for  each  two-day  period  (see  Experiment  24, 
page  587),  and  note  the  increase  in  the  daily  excretion  under  the  influence  of 
the  agar  ingestion.    What  was  the  increase  per  gram  of  agar? 

'  In  the  oxidation  process  use  10  grams  of  potassium  sulphate  instead  of  the  copper 
sulphate.     The  remainder  of  the  procedure  is  the  same  as  for  urine. 

*  More  accurate  results  will  be  secured  if  the  bacterial  nitrogen  is  determined  on  each 
individual  stool  in  the  fresh  condition. 

'Longer  periods  are  desirable  where  great  accuracy  is  desired. 


59°  PHYSIOLOGICAL   CHEMISTRY 

29.  Protein  Utilization. — By  "protein  utilization"  is  meant  the 
percentage  of  the  ingested  protein  which  is  actually  absorbed  and 
assimilated. 

This  may  be  determined  by  the  following  procedure :  Ingest  any  diet  of 
known  nitrogen  content  for  a  period  of  three  days^  (see  table,  page  569).  Collect 
all  feces  from  the  diet  making  the  "separations"  as  directed  on  page  587,  using 
carmine  as  the  initial  "marker"  and  charcoal  as  the  final  "marker"  or  vice 
versa.  Preserve  the  feces  as  directed  on  page  588.  Mix  the  total  feces  thor- 
oughly and  determine  the  nitrogen  by  the  Kjeldahl  method  (see  Chapter  XXVI  and 
note  on  page  589).  The  approximate  nitrogen  utihzation  may  be  calculated  as 
follows : 

(Food  nitrogen  —  Feces  nitrogen)  X  100 

Food^^trogen =  Approximate  percentage  nitrogen  util- 
ization. If  it  is  desired  to  ascertain  the  actual  percentage  of  the  ingested  ni- 
trogen which  has  been  utilized  by  the  body  we  must  make  a  correction  for  meta- 
boUc  nitrogen.  In  doing  this  proceed  as  follows :  Ingest  a  non-nitrogenous  diet 
as  described  on  page  589  for  a  period  of  two  days,  using  sufficient  agar-agar  to 
insure  a  daily  fecal  output  which  shall  approximate  in  weight  that  obtained  when 
the  regular  protein  diet  was  ingested.^  Separate  and  preserve  the  feces  as 
directed  on  page  588.  Mix  thoroughly  and  analyze  for  nitrogen  according  to 
the  Kjeldahl  method  (see  Chapter  XXVI  and  note  on  page  589).  Calculate  the 
actual  percentage  utihzation  of  the  diet  as  follows : 
[Food  Nitrogen  — (Fecal  nitrogen— metabolic  nitrogen)]  X 100 

^  Food~niSoi^n =^^^^^    P^'^^^*" 

age  nitrogen  utihzation.     If  urinary  nitrogen  is   determined   the  above  data 
enable  us  to  prepare  a  nitrogen  balance  (see  Experiment  34,  page  592). 

30.  Influence  of  Defective  Mastication  on  Food  Residues  in  Feces. — Rapid 
eating  accompanied  by  defective  mastication  leads  to  the  appearance  of  relatively 
large  macroscopic  food  residues  in  the  feces.  Under  some  conditions,  however,  pro- 
tein utilization  (see  above)  may  be  as  satisfactory  during  food  "bolting"  as  when 
the  food  is  very  thoroughly  masticated.^  This  problem  may  be  studied  by  the 
following  method: 

(o)  Ingest  a  diet  containing  meat  and  be  certain  to  masticate  the  diet  very 
thoroughly.  Collect  a  stool,  examine  macroscopically;  mix  carefully  and  examine 
microscopically  (see  page  229). 

{h)  Ingest  a  diet  similar  to  that  employed  in  above  experiment  (a).  "Bolt" 
the  food,  i.e.,  ingest  it  practically  without  mastication.  Examine  the  feces  as 
above.  Note  the  difference  in  the  macroscopical  and  microscopical  findings  under 
(a)  and  {b). 

If  the  nitrogen  of  food  and  feces  is  determined  we  may  calculate  the  protein 
utilization  (see  Experiment  29).  By  the  additional  determination  of  urinary 
nitrogen,  we  may  prepare  a  nitrogen  balance  (see  Experiment  34,  page  592). 

1  See  note  3,  p.  589. 

*  It  is  frequently  difficult  to  so  regulate  the  agar-agar  intake  as  to  secure  the  proper  fecal 
output.  In  such  an  event  the  proper  value  for  metabolic  nitrogen  must  be  obtained  by  cal- 
culation. For  example  if  89.1  grams  of  feces  were  excreted  per  day  on  the  protein  diet,  and 
166.5  grams  per  day  (with  a  nitrogen  value  of  0.5  gram)  when  agar  was  employed,  the 
actual  value  for  metabolic  product  nitrogen  may  be  obtained  by  the  following  proportion, 
assuming  that  the  content  of  metabolic  nitrogen  is  proportional  to  the  weight  of  feces  ex- 
creted: 89.1  :  166.5  :  -.x  :o.5.     x  =  0.268  gram  metabolic  nitrogen  per  day. 

^Foster  and  Hawk:  Jour.  Amer.  Chetn.  Soc,  37,  1347,  1915. 


METABOLISM  591 

31.  Fat  in  Feces. — A  normal  adult  will  digest  and  absorb  at  least 
90  per  cent  of  the  fat  in  the  diet  when  the  amount  ingested  does  not 
exceed  ibo  grams.  If  the  diet  contains  an  excessive  amount  of  fat, 
e.g.i  300  grams  per  day,  considerable  appears  in  the  feces.  In  pancreatic 
diseases  and  such  conditions  as  are  accompanied  by  a  decrease  in  bile 
flow  the  digestion  and  assimilation  of  fat  is  lessened. 

Experiments. — (a)  Ingest  an  ordinary  mixed  diet  containing  an  average  amount 
of  fat  per  day,  e.g.,  75-100  grams.  Collect  a  stool  and  examine  it  microscopically  as 
directed  in  Chapter  XTV.  (b)  Now  ingest  a  diet  containing  an  excessive  quantity 
of  fat,  e.g.,  300  grams  per  day.  Separate  the  feces  and  subject  a  representative 
sample  of  the  feces  from  the  high  fat  diet  to  microscopical  examination,  (c) 
If  it  is  desired  the  fat  may  be  extracted  from  some  of  the  stool  by  applying  the 
principle  involved  in  the  quantitative  determination  of  fat  in  the  Saxon  method 
(see  Chapter  XTV) .  Evaporate  the  ether  extract  and  identify  the  fat  in  the  residue 
by  tests  given  in  Chapter  IX. 

32.  Carbohydrate  in  Feces. — Under  normal  conditions  the  great 
bulk  of  the  soluble  carbohydrate  in  the  food  is  absorbed  from  the  intes- 
tine even  when  the  ingestion  is  high.  Hence  the  content  of  soluble 
carbohydrate  in  feces  is  low.     To  demonstrate  this  proceed  as  follows: 

(a)  Ingest  for  three  days  an  ordinary  mixed  diet  to  which  100  grams  of  glucose 
or  sucrose  is  daily  added.  Separate  and  preserve  the  feces  (see  page  588)  and 
when  the  final  "marker"  appears  extract  an  aUquot  portion  of  the  total  mixed  feces^ 
with  water,  decolorize  with  boneblack,  filter,  and  after  making  the  filtrate  up  to 
a  known  volimie  determine  the  sugar  by  Benedict's  method  (see  page  522). 
Calculate  the  soluble  carbohydrate  content  of  the  feces  for  the  three-day  interval. 
(b)  Proceed  as  above  with  the  exception  that  at  least  250  grams  of  sugar  should 
be  added  to  the' diet  instead  of  100  as  in  (a). 

How  did  the  daily  excretion  of  soluble  carbohydrate  in  (a)  compare  with  that 
in  (b)?  Why  is  this  so?  If  a  diet  of  known  carbohydrate  content  is  fed  this 
experiment  will  give  us  accurate  data  as  to  soluble  carbohydrate  utiUzation  (see 
Protein  Utilization,  page  590).  If  it  is  desired  this  experiment  may  be  combined 
with  the  hyperglycemia  and  glycosuria  experiments  on  pages  566  and  568.  See 
also  Experiment  36,  page  593. 

33.  Inorganic  Elements  in  the  Feces. — The  salts  of  sodium  and 
potassium  being  very  soluble  are  almost  completely  absorbed,  from  the 
intestine.  The  same  is  true  of  the  chlorides  including  that  of  sodium 
which  is  of  greatest  importance.  Hence  the  alkali  metals  and  chlorides 
are  excreted  mainly  in  the  urine  and  are  found  only  in  very  small 
amounts  in  the  feces  even  when  large  amounts  are  ingested.  With 
calcium,  magnesium,  iron  and  phosphate  conditions  are  different.  Not 
only  are  salts  of  calcium,  magnesium  and  iron  less  readily  absorbed  but 
they  are  excreted  to  a  large  extent  by  way  of  the  intestinal  mucosa  rather 
than  by  the  kidneys.     Ordinarily  about  90  per  cent  of  ingested  calcium  is 

^  If  time  permits  it  is  more  satisfactory  to  analyze  each  individual  stoolin /r«/i  condition. 


592 


PHYSIOLOGICAL   CHEMISTRY 


eliminated  by  way  of  the  feces  and  a  little  less  than  half  of  the  magnes- 
ium. From  20-30  per  cent  of  the  phosphorus  ingested  is  usually  found 
in  the  feces. 

Experiments. — (a)  Ingest  for  a  period  of  three  days  an  ordinary  mixed  diet 
without  added  salt  and  containing  no  milk.  Separate  the  feces  for  the  period  (see 
page  587)  and  retain  a  portion  of  the  well -mixed  feces  for  analysis. 

(b)  Proceed  as  above  with  the  exception  that  there  is  added  to  the  mixed  diet 
10  grams  of  conmion  salt  and  a  quart  of  milk  (containing  about  1.6  grams  of  CaO, 
0.2  gram  MgO,  1.4  grams  of  chloride  expressed  as  sodiimi  chloride,  and  2.2 
grams  P2O6).     Mix  feces  well  and  reserve  part  for  analysis. 

Ash  10  gram  samples  of  the  feces  from  the  above  diets.  Dissolve  with  the  aid 
of  a  Uttle  dilute  nitric  acid,  filter  and  make  up  to  100  c.c.  Determine  in  aliquot 
portions  of  this  solution:  (i)  Chlorides  by  Volhard  method.  (2)  Calciimi  and 
magnesivun  by  McCrudden's  method.  (3)  Phosphorus  by  uranixmi  titration. 
(For  details  of  analytical  methods  see  Chapter  XXVI.)  Calculate  the  percentages 
of  the  added  Ca,  Mg,  P,  and  CI  which  are  recovered  from  the  feces. 

For  a  more  detailed  study  of  chloride  excretion  combine  this  experiment  and 
Experiment  14  (see  Experiment  13). 


m.  METABOLISM  PROCEDURES  INVOLVING  THE 
MANIPULATION  OF  BOTH  URINE  AND  FECES 

34  Preparation  of  a  Metabolic  Balance. — This  test  entails  the 
analysis  of  the  food  ingested  and  of  the  urine  and  feces  excreted,  i.e., 
a  study  of  the  income  and  outgo.     Proceed  as  follows: 

Select  a  diet  which  is  simple,  i.e.,  consists  of  few  constituents,  and  which 
lends  itself  readily  to  accurate  chemical  analysis.  A  good  type  of  diet  for  ordi- 
nary metaboUsm  experiments  of  this  sort  consists  of  crackers  (graham  or  soda), 

BALANCE  OF  CALCIUM,  MAGNESIUM,  PHOSPHORUS,  SULPHUR,  AND  NITRO- 
GEN IN  ACROMEGALY 


Calcium 
oxide 


Magnesium  Phosphoric 
oxide        ;  anhydride  i 


Sulphur    Nitrogen 


Grams 


Ingestion  (daily) 

....      I . 404 

0.486 

3.192 

1. 190 

18.84 

Excretion  (urine) 

....      O.IS9  j 

0.160 

1.701 

1.006 

17.60 

Excretion  (feces) 

....'     1.093 

0.226 

1 .002 

0.13s 

1. 10 

Excretion  (total) , 


1.252         0.386 


2.703  I. 141        18.70 


Retention  (daily). 


0.242         0.100 


0.489         0.049         0.14 


Retention  (per  cent). 


16.2  20.6 


iS-3 


41 


0.7 


METABOLISM  593 

milk,  butter,  water  and  agar-agar  (to  prevent  constipation).  Meat  specially 
prepared  in  quantity  sufficient  for  an  entire  experiment  may  also  be  utilized. 
Ingest  uniform  quantities  of  these  dietary  constituents  each  day  for  a  period  of 
three  days.^  Make  an  accurate  collection  of  the  urine  passed  during  this  interval 
(see  page  565).  Separate  the  feces  representing  the  three-day  period  (see  page 
587),  and  analyze  foods,  urine  and  feces.  The  balances  ordinarily  prepared  are 
those  for  nitrogen,  sulphur,  phosphorus  and  calcium.  Analytical  methods  for  the 
determination  of  these  elements  may  be  found  in  Chapter  XXVI. 

The  foregoing  table  includes  balances  obtained  in  a  metaboUsm  test  on 
acromegaly.- 

35.  Excretion  of  Urinary  and  Fecal  Chloride  after  a  High  Chloride  Ingestion. — 
Combine  the  procedures  outlined  under  Experiments  14  and  33,  pages  576  and 

591- 

36.  A  Study  of  the  Elimination  of  Carbohydrate  in  Urine  and  Feces  after 

Excessive  Carbohydrate  Ingestion. — Combine  the  procedures  outlined  in  Experi- 
ments 5  and  32,  pages  568  and  591. 

1  See  note  3,  p.  589. 

^Bergeim,  Stewart  and  Hawk:  Jour.  Expl.,  Med.,  20,  218,  1914. 


38 


REAGENTS  AND  SOLUTIONS 

Alizarin.  ^ — A  i  per  cent  solution  of  alizarin  mono-sodium  sulphonate 
in  water. 

Almen's  Reagent.- — Dissolve  5  grams  of  tannic  acid  in  240  c.c.  of 
50  per  cent  alcohol  and  add  10  c.c.  of  25  per  cent  acetic  acid. 

Aluminium  Hydroxide  Cream. ^ — To  a  i  per  cent  solution  of  ammon- 
ium alum  at  room  temperature  add  a  slight  excess  of  a  i  per  cent  solu- 
tion of  ammonium  hydroxide.  Wash  by  decantation  until  the  wash 
water  shows  only  the  faintest  trace  of  residue  on  evaporation. 

Ammoniacal  Silver  Solution.* — Dissolve  26  grams  of  silver  nitrate 
in  about  500  c.c.  of  water,  add  enough  ammonium  hydroxide  to  redis- 
solve  the  precipitate  which  forms  upon  the  first  addition  of  the  ammon- 
ium hydroxide  and  make  the  volume  of  the  mixture  up  to  i  liter  with 
water. 

Ammoniimi  Thiocyanate  Solution.^ — This  solution  is  made  of  such 
a  strength  that  i  c.c.  of  it  is  equal  to  i  c.c.  of  the  standard  silver  nitrate 
solution  mentioned  below.  To  prepare  the  solution  dissolve  12.9  grams 
of  ammonium  thiocyanate,  NH4SCN,  in  a  little  less  than  a  liter  of 
water.  In  a  small  flask  place  20  c.c.  of  the  standard  sUver  nitrate 
solution,  5  c.c.  of  a  cold  saturated  solution  of  ferric  alum  and  4  c.c.  of 
nitric  acid  (sp.  gr.  1.2),  add  water  to  make  the  total  volume  100  c.c,  and 
thoroughly  mix  the  contents  of  the  flask.  Now  run  in  the  ammonium 
thiocyanate  solution  from  a  burette  until  a  permanent  red-brown  tinge  is 
produced.  This  is  the  end-reaction  and  indicates  that  the  last  trace 
of  silver  nitrate  has  been  precipitated.  Take  the  burette  reading  and 
calculate  the  amount  of  water  necessary  to  use  in  diluting  the  ammon- 
ium thiocyanate  in  order  that  10  c.c.  of  this  solution  may  be  exactly 
equal  to  10  c.c.  of  the  silver  nitrate  solution.  Make  the  dilution  and 
titrate  again  to  be  certain  that  the  solution  is  of  the  proper  strength. 

Amold-Lipliawsky  Reagent." — This  reagent  consists  of  two  definite 
solutions  which  are  ordinarily  preserved  separately  and  mixed  just  before 
using.     The  two  solutions  are  prepared  as  follows: 

{a)  One  per  cent  aqueous  solution  of  potassium  nitrate. 

^  Indicator  in  various  procedures,  pp.  174  and  481. 

-Ott's  precipitation  test,  p.  429.     Determinatiofi  of  lactalbumin,  p.  329. 

^  Removal  of  protein  in  various  methods,  pp.  329,  485. 

*  Salkowski's  method,  p.  516. 

*  Volhard-Arnold  method,  p.  556,  and  Dehn-'Clark  method,  p.  557. 
'  Arnold-Lipliawsky  reaction,  p.  440. 

594 


REAGENTS    AND    SOLUTIONS  595 

(b)  One  gram  of  ^-amino-acetophenon  dissolved  in  loo  c.c.  of 
distilled  water  and  enough  hydrochloric  acid  (about  2  c.c.)  added  drop 
by  drop,  to  cause  the  sdution,  which  is  at  first  yellow,  to  become  entirely 
colorless.     An  excess  of  acid  must  be  avoided. 

Asbestos  for  Suction  Filters.^  The  asbestos  is  shredded,  placed  in  a 
wide  mouth  flask  and  covered  with  10  per  cent  HCl.  Heat  on  water- 
bath  for  five  hours.  Filter  on  Buchner  funnel,  wash  free  from  acid, 
return  to  the  flask,  cover  with  5  per  cent  NaOH  and  heat  on  water-bath 
for  three  hours.  Filter,  wash  free  from  alkali,  then  with  dilute  acid 
and  finally  with  water  until  free  from  acid.  Suspend  in  a  large  volume 
of  water,  allow  to  settle  for  five  minutes.  Pour  ofi"  the  upper  two- thirds 
and  discard.  Repeat  the  washing  of  the  desired  coarse  portion  several 
times  until  the  supernatant  liquid  remains  nearly  clear. 

Bang's  Sugar  Reagents.- — (a)  Acid  KCl  Solution. — Consisting 
of  1360  c.c.  of  saturated  KCl  to  which  is  added  640  c.c.  of  water  and 
1.5  c.c.  25  per  cent.  HCl. 

{h)  Stock  Copper  Solution. — Introduce  into  a  1000  c.c.  flask  700  c.c. 
of  boiled  and  cooled  water.  Warm  to  about  3o°C.  and  add  160  grams 
of  pure  potassium  bicarbonate  in  powder  form.  When  dissolved  and 
66  grams  of  pure  KCl.  Cool  and  then  add  100  grams  potassium  carbon- 
ate. Finally  add  100  c.c.  of  4.4.  per  cent,  solution  of  pure  crystalline 
copper  sulphate.  Let  stand  a  short  time,  then  make  to  mark  with 
boiled  water.     Allow  to  stand  a  day  or  so  before  using. 

(c)  N/200  I  Solution. — Made  fresh  each  day.  Dilute  N/io  I 
solution  20  times.  Or  make  as  follows:  Introduce  into  a  100  c.c. 
flask  2  grams  KI,  1-2  c.c.  of  2  per  cent  KIO3  solution  and  5  c.c.  of  N/io 
HCl.     Make  to  mark  with  boiled  and  cooled  distilled  water. 

{d)  Starch  Solution. — A  i  per  cent,  solution  of  Kahlbaum's  soluble 
starch  in  a  saturated  KCl  solution. 

(e)  Dilute  Copper  Solution. — Dilute  300  c.c.  of  the  stock  solution 
to  1000  c.c.  Mix  with  only  gentle  shaking.  Let  stand  several  hours 
before  using. 

Barfoed's  Solution.'' — Dissolve  4.5  grams  of  neutral,  crystalUzed 
copper  acetate  in  100  c.c.  of  water  and  add  1.2  c.c.  of  50  per  cent  acetic 
acid. 

Baryta  Mixture.'' — A  mixture  consisting  of  i  volume  of  a  saturated 
solution  of  bariuyi  nitrate  and  2  volumes  of  a  saturated  solution  of 
barium  hydroxide. 

'  See  methods  entailing  use  of  Gooch  crucibles. 
-  Determination  of  sugar,  pages  280  and  524. 
^  Barfoed's  test,  p.  30. 
*  Isolation  of  urea  from  urine,  p.  375. 


596  PHYSIOLOGICAL   CHEMISTRY 

Basic  Lead  Acetate  Solution.  ^^ — This  solution  possesses  the  following 

formula: 

Lead  acetate ♦ 180  grams. 

Lead  oxide  (Litharge) no  grams. 

Distilled  water  to  make 1000  grams. 

Dissolve  the  lead  acetate  in  about  700  c.c.  of  distilled  water,  with  boiling. 
Add  this  hot  solution  to  the  finely  powdered  lead  oxide  and  boil  for  one- 
half  hour  with  occasional  stirring.  Cool,  filter  and  add  sufficient  dis- 
tilled water  to  the  filtrate  to  make  the  weight  i  kg. 

Benedict's  Solutions. ^ — First  Modification. — Benedict's  modified 
Fehling  solution  consists  of  two  definite  solutions — a  copper  sulphate 
solution  and  an  alkaline  tartrate  solution,  which  may  be  prepared  as 
follows : 

Copper  sulphate  solution  =  34.65  grams  of  copper  sulphate  dissolved 
in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =  100  grams  of  anhydrous  sodium  carbon- 
ate and  173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to 
100  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done 
to  prevent  deterioration. 

Second  Modification. — Benedict  has  further  modified  his  solution 
and  has  succeeded  in  obtaining  one  which  does  not  deteriorate  upon 
long  standing.     It  has  the  following  composition: 

Copper  sulphate 17.3  grams. 

Sodium  citrate i73-o  grams. 

Sodium  carbonate 100. o  grams. 

Distilled  water  to  make  i  liter. 

With  the  aid  of  heat  dissolve  the  sodium  citrate  and  carbonate  in 
about  600  c.c.  of  water.  Pour  (through  a  folded  filter  paper  if  neces- 
sary) into  a  glass  graduate  and  make  up  to  850  c.c.  Dissolve  the 
copper  sulphate  in  about  100  c.c.  of  water  and  make  up  to  150  c.c. 
Pour  the  carbonate-citrate  solution  into  a  large  beaker  or  casserole  and 
add  the  copper  sulphate  solution  slowly,  with  constant  stirring.  The 
mixed  solution  is  ready  for  use  and  does  not  deteriorate  upon  long 
standing. 

Benedict's  Sugar  Reagent.^ 

Copper  sulphate  (crystallized) 18.0  grams. 

Sodium  carbonate  (crystallized,  one-half  the  weight  of  the 

anhydrous  salt  may  be  used) 200.0  grams. 

Sodium  or  potassium  citrate 200.0  grams. 

Potassium  thiocyanate 125.0  grams. 

Potassium  ferrocyanide  (5  per  cent  solution). 5.0  c.c. 

Distilled  water  to  make  a  total  volume  of 1000. o  c.c. 

^  Indican  determination,  p.  542. 

^Benedict's  modifications  of  Fehling's  test,  pp.  27  and  417. 

^  Quantitative  determination  of  sugar,  p.  522. 


REAGENTS    AND    SOLUTIONS  597 

With  the  aid  of  heat  dissolve  the  carbonate,  citrate  and  thiocyanate 
in  enough  water  to  make  about  800  c.c.  of  the  mixture  and  filter  if 
necessary. 

Dissolve  the  copper  sulphate  separately  in  about  100  c.c.  of  water 
and  pour  the  solution  slowly  into  the  other  liquid,  with  constant  stirring. 
Add  the  ferrocyanide  solution,  cool  and  dilute  to  exactly  i  liter.  Of  the 
various  constituents,  the  copper  salt  only  need  be  weighed  with  exact- 
ness.    Twenty-five  c.c.  of  the  reagent  are  reduced  by  50  mg.  of  glucose. 

Benedict's  Sulphur  Reagent. 

Crystallized  copper  nitrate,  sulphur-free  or  of  known  sulphur 

content 200  grams. 

Sodium  or  potassium  chlorate 50  grams. 

Distilled  water  to 1000  c.c. 

Benzidine  Solutions  for  Volumetric  Sulphur  Determinations. 

— (a)  Rosenheim  and  Drummond. — Rub  4  grams  of  benzidine  (Kahl- 
baum)  into  a  fine  paste  with  about  10  c.c.  of  water  and  transfer  to  a 
2-liter  flask  with  the  aid  of  about  500  c.c.  of  water.  Add  500  c.c.  of  con- 
centrated HCl  (sp.  gr.  1. 1 9)  and  make  up  to  2  liters  with  distilled 
water.  150  c.c.  of  this  solution,  which  keeps  indefinitely,  are  sufficient 
to  precipitate  o.i  gram  H2SO4. 

{h)  Raiziss  and  Duhin. — Put  6.7  grams  of  benzidine  (Merck  Reagent) 
in  a  i-liter  flask,  add  29  c.c.  of  hydrochloric  acid  (sp.  gr.  1.12)  and  dilute 
up  to  the  mark. 

Bertrand  Sugar  Reagents.^ — {a)  Copper  Sulphate  Solution. — Forty 
grams  of  pure  crystallized  copper  sulphate  are  dissolved  in  water  to 
make  a  liter. 

(&)  Dissolve  200  grams  of  Rochelle  salts  and  150  grams  of  NaOH  in 
water  to  make  1000  c.c. 

(c)  Acid  Ferric  Sulphate  Solution. — Dissolve  50  grams  of  ferric  sul- 
phate in  about  200  c.c.  of  water  and  pour  into  this  a  mixture  of  200  c.c. 
of  concentrated  sulphuric  acid  diluted  with  about  400  c.c.  of  water. 
Mix  and  make  to  1000  c.c. 

{d)  Potassium  Permanganate  Solution. — Dissolve  5  grams  of  potas- 
sium permanganate  in  water  to  make  1000  c.c.  Standardization. — 
Dissolve  0.250  gram  of  ammonium  oxalate  in  50-100  c.c.  of  water  add 
1-2  c.c.  of  concentrated  sulphuric  acid  and  titrate  with  the  permangan- 
ate to  a  rose  color.  Multiply  the  number  of  grams  of  oxalate  used  by 
0.895  ^^  g^t  the  equivalent  in  Cu  of  the  number  of  cubic  centimeters  of 
permanganate  used.     Calculate  the  Cu  value  of  i  c.c. 

*  Determination  of  sugar,  p.  527. 


598  PHYSIOLOGICAL    CHEMISTRY 

Bial's  Reagent.^ 

Orcinol 1.5    grams. 

Fuming  HCl 500 .  00  grams. 

Ferric  chloride  (10  per  cent) 20-30  drops. 

Biuret  Reagent,  Gies.-— This  reagent  consists  of  lo  per  cent  KOH 
solution  to  which  25  c.c.  of  3  per  cent  CUSO4  solution  per  liter  has  been 
added.  This  imparts  a  slight  though  distinct  blue  color  to  the  clear 
liquid. 

Biuret  Paper  (Kantor  and  Gies).- — Immerse  filter  paper  in  Gies' 
Biuret  Reagent  (above)  then  dry  and  cut  into  strips. 

Black's  Reagent.^ — Made  by  dissolving  5  grams  of  ferric  chloride 
and  0.4  gram  of  ferrous  chloride  in  100  c.c.  of  water. 

Blood  Serum. — This  may  easily  be  obtained  in  quantity  by  the 
procedure  described  under  Hemagglutination  in  the  chapter  on  Blood. 

Boas'  Reagent."* — Dissolve  5  grams  of  resorcinol  and  3  grams  of 
sucrose  in  100  c.c.  of  50  per  cent  alcohol. 

Bonnano's  Reagent. — Dissolve  2  grams  of  sodium  nitrite  in  100  c.c. 
of  concentrated  hydrochloric  acid. 

Bottu's  Reagent. — To  3.5  grams  of  o-nitrophenylpropiolic  acid 
add  5  c.c.  of  a  freshly  prepared  10  per  cent  solution  of  sodium  hydroxide 
and  make  the  volume  of  the  solution  i  liter  with  distilled  water. 

Carmine -Fibrin.^ — Prepared  by  running  fibrin  through  a  meat 
chopper,  washing  carefully  and  placing  in  3^^  per  cent  ammoniacal 
carmine  solution  (very  little  excess  ammonia  should  be  present)  until 
the  maximum  coloration  of  the  fibrin  (dark  red)  is  obtained.  The  fibrin 
is  then  washed  in  water  and  in  water  acidified  with  acetic  acid.  It  is 
preserved  under  glycerol. 

Chloride  Reagents  for  Blood  Analysis.^— (a)  An  acid  M/29.25 
solution  of  silver  nitrate,  i  c.c.  of  which  is  equivalent  to  2  mg.  of  NaCl. 

AgNOs 5.812  grams. 

HN03(sp.  gr.  1.42) 250  c.c. 

Water  to 1000  c.c. 

{b)  A  solution  of  M/58.5  potassium  iodide,  i  c.c.  of  which  is  equiva- 
lent to  I  mg.  of  NaCl. 

KI 3.0  grams. 

Water  to 1000  c.c. 

This  solution  is  standardized  against  the  silver  solution  by  adding  5  c.c. 
of  the  latter  to  5  c.c.  of  solution  (c)  and  titrating  with  the  iodide  solution 

I  Test  for  pentose,  p.  37. 

*  Protein  tests,  p.  100. 

'Black's  reaction,  p.  441. 

♦Test  for  free  acid,  p.  154- 

"Tests  on  proteases,  p.  12. 

«  Method  of  McLean  and  Van  Slyke,  p.  286. 


REAGENTS    AND    SOLUTIONS  599 

to  the  blue  end  point.     The  iodide  solution  is  then  diluted  to  such  a 
degree  that  lo  c.c.  are  exactly  equivalent  to  5  c.c.  of  the  silver  solution. 
(c)  A  solution  used  as  an  indicator,  to  regulate  acidity,  and  provide 
an  oxidizing  agent. 

Sodium  citrate  (NaaCoHsOz+sM  H2O) 446.0  grams. 

Sodium  nitrite 20.0  grams. 

Soluble  starch 2.5  grams. 

Water  to looo.o  c.c. 

The  starch  is  first  dissolved  with  the  aid  of  heat  in  about  500  c.c.  of  the 
water.  The  citrate  and  nitrite  are  then  added,  and  the  mixture  is  heated 
until  all  is  dissolved.  The  solution  while  still  hot  is  filtered  through 
cotton,  the  filter  washed  with  hot  water,  the  filtrate  allowed  to  cool, 
and  made  up  to  1000  c.c.     The  solution  keeps  indefinitely. 

Combined  Hydrochloric  Acid  (Protein  Salt). — To  prepare  so-called 
combined  hydrochloric  acid  simply  add  a  soluble  protein  such  as  Witte's 
peptone  to  free  hydrochloric  acid  of  the  desired  strength  until  it  no 
longer  responds  to  free  acid  tests  (see  chapter  on  Gastric  Digestion). 
For  example,  if  0.2  per  cent  combined  acid  is  required  the  protein  would 
be  added  to  0.2  per  cent  free  hydrochloric  acid. 

Strictly  speaking  there  is  no  such  thing  as  "combined"  acid  in  this 
sense.  When  the  protein  is  added  a  protein  salt  of  the  acid  is  formed 
which  ionizes  differently  from  the  free  acid. 

Congo  Red.^ — Dissolve  0.5  gram  of  Congo  red  in  90  c.c.  of  water 
and  add  10  c.c.  of  95  per  cent  alcohol. 

Congo  Red-Fibrin. — This  may  be  prepared  by  placing  fibrin  in 
faintly  alkaline  Congo  red  solution  and  heating  to  8o°C.  The  fibrin  is 
then  washed  and  preserved  under  glycerol. 

Creatinine,  Standard  Solution  for  Colorimetric  Method. ^ — Dissolve 
I  gram  of  pure  creatinine  in  1000  c.c.  of  N/io  HCl.  The  solution  con- 
tains I  mg.  of  creatinine  per  cubic  centimeter. 

Cross  and  Sevan's  Reagent. — Combine  t-d'o  parts  of  concentrated 
hydrochloric  acid  and  one  part  of  zinc  chloride  hy  weight. 

Ehrlich's  Diazo  Reagent. '' — Two  separate  solutions  should  be  pre- 
pared and  mixed  in  definite  proportions  when  needed  for  use. 

(a)  Five  grams  of  sodium  nitrite  dissolved  in  i  liter  of  distilled  water. 

{h)  Five  grams  of  sulphanilic  acid  and  50  c.c.  of  hydrochloric  acid  in 
I'liter  of  distilled  water. 

Solutions  (a)  and  {b)  should  be  preserved  in  well-stoppered  vessels 
and  mixed  in  the  proportion  i :  50  when  required.     Green  asserts  that 

^  Test  for  free  acid,  p.  153. 

^  Determination  of  creatinine,  p.  506. 

'  Ehrlich's  diazo  reaction,  p.  454. 


6oo  PHYSIOLOGICAL   CHEMISTRY 

greater  delicacy  is  secured  by  mixing  the  solutions  in  the  proportion 
I  :ioo.  The  sodium  nitrite  deteriorates  upon  standing  and  becomes 
unfit  for  use  in  the  course  of  a  few  weeks. 

Esbach's  Reagent.^ — Dissolve  lo  grams  of  picric  acid  and  20  grams 
of  citric  acid  in  i  liter  of  water, 

Fehling's  Solution.^ — Fehling's  solution  is  composed  of  two  definite 
solutions — a  copper  sulphate  solution  and  an  alkaline  tartrate  solution, 
which  may  be  prepared  as  follows: 

Copper  sulphate  solution  =  34.65  grams  of  copper  sulphate  dissolved 
in  water  and  made  up  to  500  c.c. 

Alkaline  tartrate  solution  =125  grams  of  potassium  hydroxide  and 
173  grams  of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  c.c. 

These  solutions  should  be  preserved  separately  in  rubber-stoppered 
bottles  and  mixed  in  equal  volumes  when  needed  for  use.  This  is  done 
to  prevent  deterioration. 

Ferric  Alum  Solution. ^ — A  cold  saturated  solution. 

Folin-Shaffer  Reagent.'*- — This  reagent  consists  of  500  grams  of  am- 
monium sulphate,  5  grams  of  uranium  acetate,  and  60  c.c.  of  10  per 
cent  acetic  acid  in  650  c.c.  of  distilled  water. 

Formalin  Solution  (Neutral).^ — To  50  c.c.  of  commercial  formalde- 
hyde solution  (30-40  per  cent)  add  i  c.c.  of  phenolphthalein  solution 
and  then  standard  alkali  solution  until  the  mixture  assumes  a  faint 
red  color.  The  solution  should  be  freshly  prepared  for  each  set  of 
determinations.         * 

Fiurfural  Solution.^ — Add  i  c.c.  of  furfural  to  1000  c.c.  of  distilled 
water. 

Gallic  Acid  Solution.^ — A  saturated  alcoholic  solution. 

Guaiac  Solution.^ — Dissolve  0.5  gram  of  guaiac  resin  in  30  c.c.  of 
95  per  cent  alcohol. 

Gulick's  Acid  Oxidizing  Mixture.^ — To  125  c.c.  of  ammonia  free 
water  add  40  c.c.  of  sulphuric  acid,  5  c.c.  of  a  saturated  solution  of 
mercuric  chloride,  and  20  grams  of  potassium  sulphate.  Then  make  up 
to  200  c.c.  with  ammonia-free  water. 

Gulick's  ModifiedWinkler Solution." — Dissolve  40  grams  of  sodium 

'  Esbach's  method,  p.  532. 

2  Fehling's  method,  p.  523.     Fehling's  test,  pp.  26  and  416. 

^  Volhard-Arnold  method,  p.  556. 

*  Folin-Shaffer  method,  p.  511. 

'  Formol  titration  procedure,  p.  502. 

«  Mylius's  modification  of  Pettenkofer's  test,  pp.  208  and  434.  v.  Udrdnsky's  test,  pp. 
208  and  434. 

^  Gallic  acid  test,  p.  32  3. 

*  Guaiac  test,  pp.  15,  235,  262,  and  432. 

*  Determination  of  total  nitrogen,  p.  490. 
'0  Determination  of  total  nitrogen,  p.  490. 


REAGENTS    ANT)    SOLUTIONS  6oi 

hydroxide  in  about  200  c.c.  of  ammonia-free  water.  Mix  15  grams  of 
mercuric  iodide  and  10  grams  of  potassium  iodide  and  dissolve  in  about 
15  c.c.  of  water.  Transfer  with  the  aid  of  the  alkali  to  a  500  c.c.  volu- 
metric flask  and  make  up  to  500  c.c.  with  ammonia-free  water.  Trans- 
fer to  an  Erlenmeyer  flask  and  let  stand  24  hours  to  settle. 

Giinzberg's  Reagent.^ — Dissolve  2  grams  of  phloroglucinol  and  i 
gram  of  vanillin  in  100  c.c.  of  95  per  cent  alcohol. 

Haines'  Solution.- — This  solution  may  be  prepared  by  dissolving 
8.314  grams  of  copper  sulphate  in  400  c.c.  of  water  adding  40  c.c.  of 
glycerol  and  500  c.c.  of  5  per  cent  potassium  hydroxide  solution. 

Hammarsten's  Reagent.^ — Mix  i  volume  of  25  per  cent  nitric 
acid  and  19  volumes  of  25  per  cent  hydrochloric  acid  and  add  i  volume 
of  this  acid  mixture  to  4  volumes  of  95  per  cent  alcohol.  It  is  prefer- 
able that  the  acid  mixture  be  prepared  in  advance  and  allowed  to  stand 
until  yellow  in  color  before  adding  it  to  the  alcohol. 

Hayem's  Solution. — This  solution  has  the  following  formula: 

Mercuric  chloride 0.25  grams. 

Sodium  chloride 0.5    grams. 

Sodium  sulphate 2.5    grams. 

Distilled  water 100. o    grams. 

Hopkins-Cole  Reagent.'' — To  i  liter  of  a  saturated  solution  of 
oxalic  acid  add  60  grams  of  sodium  amalgam  and  allow  the  mixture 
to  stand  until  the  evolution  of  gas  ceases.  Filter  and  dilute  with  2-3 
volumes  of  water. 

Hopkins-Cole  Reagent  (Benedict's  Modification). — Ten  grams 
of  powdered  magnesium  are  placed  in  a  large  Erlenmeyer  flask  and 
shaken  up  with  enough  distilled  water  to  liberally  cover  the  magnesium. 
Two  hundred  and  fifty  c.c.  of  a  cold,  saturated  solution  of  oxalic  acid  is 
now  added  slowly.  The  reaction  proceeds  very  rapidly  and  with  the 
liberation  of  much  heat,  so  that  the  flask  should  be  cooled  under  running 
water  during  the  addition  of  the  acid.  The  contents  of  the  flask  are 
shaken  after  the  addition  of  the  last  portion  of  the  acid  and  then  poured 
upon  a  filter,  to  remove  the  insoluble  magnesium  oxalate.  A  little 
wash  water  is  poured  through  the  filter,  the  filtrate  acidified  with 
acetic  acid  to  prevent  the  partial  precipitation  of  the  magnesium  on  long 
standing,  and  made  up  to  a  liter  with  distilled  water.  This  solution 
contains  only  the  magnesium  salt  of  glyoxylic  acid. 

Hypobromite  Solution.^ — The  ingredients  of  this  solution  should 

'  Test  for  free  acid,  p.  154. 

-Haines'  test,  p.  419. 

^  Hammarsten's  reaction,  pp.  207  and  433. 

*  Hopkins-Cole  reaction,  p.  98. 

*  Methods  for  determination  of  urea,  p.  496. 


602  PHYSIOLOGICAL   CHEMISTRY 

be  prepared  in  the  form  of  two  separate  solutions  which  may  be  united 
as  needed. 

(a)  Dissolve  125  grams  of  sodium  bromide  in  water,  add  125  grams 
of  bromine  and  make  the  total  volume  of  the  solution  i  liter. 

{h)  A  solution  of  sodium  hydroxide  having  a  specific  gravity  of 
1.25,     This  is  approximately  a  22.5  per  cent  solution. 

Preserve  both  solutions  in  rubber-stoppered  bottles  and  when  needed 
for  use  mix  i  volume  of  solution  (a),  i  volume  of  solution  (6),  and  3 
volumes  of  water. 

Iodine  Solution  (N/io).^ — Weigh  out  12.685  grams  of  pure  resub- 
limed  iodine  into  a  small  weighing  bottle  using  a  porcelain  spatula. 
Dissolve  18  grams  of  pure  KI  in  about  150  c.c.  of  water.  Transfer  the 
iodine  to  a  liter  flask  washing  out  the  last  traces  with  some  of  the  KI 
solution,  which  is  then  poured  into  the  flask.  Stopper  and  shake 
occasionally  until  dissolved.  If  necessary  a  few  more  crystals  of  KI 
may  be  added  to  aid  solution.  Dilute  to  the  mark  and  mix  well. 
Keep  in  glass-stoppered  bottle  in  cool  dark  place.  Standardize  at  once 
against  N/io  sodium  thoisulphate  solution.  Measure  out  accurately 
25  c.c.  of  the  iodine  solution  into  an  Erlenmeyer  flask,  run  in  sodium 
thiosulphate  until  the  color  is  pale  yellow,  then  add  a  few  cubic  centi- 
meters of  a  I  per  cent  solution  of  starch  (preferably  soluble  starch) 
and  titrate  to  disappearance  of  blue  color.  Care  should  be  taken  near 
the  end  point. 

Iodine  Solution. ^ — Prepare  a  2  per  cent  solution  of  potassium  iodide 
and  add  suflicient  iodine  to  color  it  a  deep  yellow. 

Iodine-Zinc  Chloride  Reagent.^ — Dissolve  20  grams  of  zinc  chloride 
in  8.5  c.c.  of  water.  Cool,  and  introduce  iodine  solution  (3  grams  KI+ 
1.5  gram  I  in  60  c.c.  of  water)  drop  by  drop  until  iodine  begins  to  pre- 
cipitate. 

Kraut's  Reagent.^ — Dissolve  272  grams  of  potassium  iodide  in 
water  and  add  80  grams  of  bismuth  subnitrate  dissolved  in  200  grams 
of  nitric  acid  (sp.  gr.  1.18).  Permit  the  potassium  nitrate  to  crystallize 
out.  then  filter  it  off  and  make  the  filtrate  up  to  i  liter  with  water. 

Lead  Acetate,  Basic.     (See  Basic  Lead  Acetate.) 

Lugol's  Solution.^ — Dissolve  4  grams  of  iodine  and  6  grams  of  potas- 
sium iodide  in  100  c.c.  of  distilled  water. 

Magnesia  Mixture.^ — Dissolve  175  grams  of  magnesium  sulphate 
and  350  grams  of  ammonium  chloride  in  1400  c.c.  of  distilled  water. 

'  Determination  of  acetone  and  acetoacetic  acid,  p.  5  33. 

*  Iodine  test,  p.  45. 

^  Amyloid  formation,  p.  49. 

*  Rosenheim's  bismuth  test  for  choline,  p.  357. 

'  Gunning's  iodoform  test,  page  436,  and  Bardach's  reaction,  p.  loi. 
6  Method  for  determination  of  total  phosphorus,  p.  554. 


REAGENTS    AND    SOLUTIONS  603 

Add  700  grams  of  concentrated  ammonium  hydroxide,  mix  thoroughly, 
and  preserve  the  mixture  in  a  glass-stoppered  bottle. 

Magnesium  Nitrate  Solution  for  Ignition.^ — Dissolve  320  grams  of 
calcined  magnesia  in  nitric  acid,  avoiding  an  excess  of  the  latter;  then 
add  a  little  calcined  magnesia  in  excess;  boil;  filter  from  the  excess  of 
magnesia,  ferric  oxide  etc.,  and  dilute  with  water  to  2  liters. 

Methyl  Red.- — Saturated  solution  in  50  per  cent  alcohol. 

Millon's  Reagent."* — Digest  i  part  (by  weight)  of  mercury  with 
2  parts  (by  weight)  of  nitric  acid  (sp.  gr.  1.42)  and  dilute  the  resulting 
solution  with  2  volumes  of  water. 

Molisch's  Reagent.^ — A  15  per  cent  alcoholic  solution  of  a-naphthol. 

Molybdate  Solution.^ — Dissolve  100  grams  of  molybdic  acid  in  144 
c.c.  of  ammonium  hydroxide  (sp.  gr.  0.90)  and  271  c.c.  of  water;  slowly 
and  with  constant  stirring  pour  the  solution  thus  obtained  into  489  c.c. 
of  nitric  acid  (sp.  gr.  1.42)  and  1148  c.c.  of  water.  Keep  the  mLxture  in 
a  warm  place  for  several  days,  or  until  a  portion  heated  to  4o°C.  deposits 
no  yellow  precipitate  of  ammonium  phosphomolybdate.  Decant  the 
solution  from  any  sediment  and  preserve  in  glass-stoppered  bottles. 

Momer's  Reagent.'^ — Thoroughly  mix  i  volume  of  formalin,  45 
volumes  of  distilled  water,  and  55  volumes  of  concentrated  sulphuric 
acid. 

Nakayama's  Reagent.'' — Prepared  by  combining  99  c.c.  of  alcohol 
and  I  c.c.  of  fuming  hydrochloric  acid  containing  4  grams  of  ferric 
chloride  per  liter. 

a-Naphthol  Solution.^ — Dissolve  i  gram  of  a-naphthol  in  100  c.c  of 
95  per  cent,  alcohol. 

Nessler-Winkler  Solution. 

Mercuric  iodide lo  grams. 

Potassium  iodide 5  grams. 

Sodium  hydroxide 20  grams. 

Water 100  c.c. 

The  mercuric  iodide  is  rubbed  up  in  a  small  porcelain  mortar  with 
water,  then  washed  into  a  flask  and  the  potassium  iodide  added.  The 
sodium  hydroxide  is  dissolved  in  the  remaining  water  and  the  cooled 
solution  added  to  the  above  mixture.  The  solution  cleared  by  standing 
is  preserved  in  a  dark  bottle. 

'  Determination  of  phosphorus,  p.  554. 

^  Determination  of  H  ion  concentration,  pp.  158  and  480. 

'  Millon's  reaction,  p.  97. 

*  Molisch's  reaction,  p.  21. 

*  Detection  and  determination  of  phosphorus,  pp.  129  and  554. 
'  Momer's  test,  p.  86. 

^  Nakayama's  reaction,  pp.  207  and  433. 

*  Oxidases  p.  14.     For  other  a-naphthol  solution  see  Molisch  reaction. 


6o4  PHYSIOLOGICAL   CHEMISTRY 

Neutral  Olive  Oil.^ — Shake  ordinary  olive  oil  with  a  lo  per  cent 
solution  of  sodium  carbonate,  extract  the  mixture  with  ether,  and 
remove  the  ether  by  evaporation.     The  residue  is  neutral  olive  oil. 

Neutral  Red.^^ — ^A  i  per  cent  solution  in  50  per  rent  alcohol. 

p-Nitrophenol.^ — A  i  per  cent  solution  in  50  per  cent  alcohol. 

Nylander's  Reagent.^ — Digest  2  grams  of  bismuth  sub  nitrate 
and  4  grams  of  Rochelle  salt  in  100  c.c.  of  a  10  per  cent  solution 
of  potassium  hydroxide.  The  reagent  should  then  be  cooled  and 
filtered. 

Obennayer's  Reagent.^ — Add  2-4  grams  of  ferric  chlorid  to  a 
liter  of  hydrochloric  acid  (sp.  gr.  1.19). 

Oxalated  Plasma.^ — Allow  arterial  blood  to  run  into  an  equal  volume 
of  0.2  per  cent  ammonium  oxalate  solution. 

Para-dimethylaminobenzaldehyde  Solution.^ — This  solution  is  made 
by  dissolving  5  grams  of  para-dimethylaminobenzaldehyde  in  100  c.c. 
of  10  per  cent  sulphuric  acid. 

Para-phenylenediamine  Hydrochloride  Solution.'^ — Two  grams  dis- 
solved in  100  c.c.  of  water. 

Peters'  Sugar  Reagents.^ — {a)  Copper  Solution. — Dissolve  34.639 
grams  of  highest  purity  crystallized  copper  sulphate  (such  as  Kahl- 
baum's  "zur  analyse  mit  garantieschein")  in  water  to  make  500  c.c. 

{h)  Alkaline  Tartrate  Solution. — Dissolve  173  grams  of  sodium 
potassium  tartrate  and  125  grams  of  potassium  hydroxide  in  water  to 
make  500  c.c. 

(c)  iV/5  Sodium  Thiosulphate. — Dissolve  about  50  grams  of  ordinary 
c.p.  sodium  thiosulphate  or  exactly  49.66  grams  of  the  pure,  dry, 
recrystallized  salt,  in  enough  boiled  out  distilled  water  to  make  a 
liter.  Allow  to  stand  for  several  days.  The  solution  should  be  stan- 
dardized against  the  copper  solution  prepared  as  above.  For  this 
purpose  introduce  20  c.c.  of  the  copper  solution  into  a  200  c.c.  Erlen- 
meyer  flask,  add  20  c.c.  of  strong  acetic  acid  (30  per  cent)  and  40  c.c.  of 
water.  Add  about  7  grams  of  a  saturated  solution  of  KI  and  titrate 
with  the  thiosulphate  using  starch  as  an  indicator.  Calculate  the 
equivalent  of  i  c.c.  of  thiosulphate  in  Cu.  One  c.c.  of  the  copper 
sulphate  solution  contains  17.647  mg.  of  Cu.  The  thiosulphate  remains 
constant  for  some  months.     It  should  be  kept  in  a  dark  bottle. 

*  Emulsification  of  fats,  p.  180. 

'^  Determination  of  H  ion  concentration,  pp,  158  and  480. 
'Nylander's  test,  pp.  29  and  420. 

*  Obermayer's  test,  p.  388. 

^  Experiments  on  blood  plasma,  p.  268. 

*  Herter's  para-dimethylaminobenzaldehyde  reaction,  p.  219. 
'  Detection  of  hydrogen  peroxide,  p.  323. 

*  Determination  of  sugar,  p.  525. 


REAGENTS   AND    SOLUTIONS  605 

Phenolphthalein.^ — Dissolve  i  gram  of  phenolphthalein  in  loo  c.c. 
of  95  per  cent  alcohol. 

Permanganate  Solution  (Alkaline)  for  Van  Slyke  Method.  ^ — The 

alkaline  permanganate  solution  contains  50  grams  of  potassium  per- 
manganate and  25  grams  of  potassium  hydroxide  per  liter. 

Potassium  Permanganate  Standard  (N/io)  Solution. — Dissolve 
3.162  grams  of  pure  potassium  permanganate  in  a  liter  of  distilled 
water,  allow  to  stand  a  few  days,  and  filter  through  glass  wool.  Stand- 
ardize against  N/io  oxalic  acid  solution  or  against  pure  dry  sodium  or 
potassium  oxalate.  One  c.c.  of  N/io  permanganate  is  equivalent  to 
7.0  mg.  of  sodium  oxalate. 

Phenylhydrazine  Mixture.^ — This  mixture  is  prepared  by  com- 
bining I  part  of  phenylhydrazine-hydrochloride  and  2  parts  of  sodium 
acetate  by  weight.     These  are  thoroughly  mixed  in  a  mortar. 

Phenylhydrazine -Acetate  Solution.^ — This  solution  is  prepared  by 
mixing  i  volume  of  glacial  acetic  acid,  i  volume  of  water,  and  2  volumes 
of  phenylhydrazine  (the  base). 

Picramic  Acid. — Permanent  Standard  for  Lewis-Benedict  Blood  Sugar 

Method} — A   solution   of   picramic   acid   makes   a   very   satisfactory 

permanent  standard.     The  color  is  identical  in  quality  with  that  formed 

in  the  method  and  its  solution  keeps  perfectly.     The  formula  of  the 

permanent  standard  is: 

Picramic  acid o .  064  gram. 

Sodium  carbonate  (anhydrous) *. o.  loo  gram. 

Water  to  make 1000. o      c.c. 

Dissolve  the  picramic  acid  with  the  aid  of  heat  in  25-50  c.c.  of  distilled 
water  which  has  been  made  alkaline  with  sodium  carbonate.  Cool  and 
dilute  to  I  liter.  This  solution  has  the  same  intensity  of  color  as  that 
obtained  by  the  proposed  method  with  0.64  mg.  of  sugar  when  the 
final  volume  of  the  reaction  fluid  is  made  10  c.c.  The  solution  should 
be  standardized  against  pure  glucose.  A  satisfactory  preparation  of 
picramic  acid  may  be  obtained  from  the  J.  T.  Baker  Chemical  Co., 
Phillipsburg,  N.  J. 

Roberts'  Reagent.*' — Mix  i  volume  of  concentrated  nitric  acid  and 
5  volumes  of  a  saturated  solution  of  magnesium  sulphate. 

Rosenheim's  lodo-Potassiimi  Iodide  Solution." — Dissolve  2  grams 
of  iodine  and  6  grams  of  potassium  iodide  in  100  c.c.  of  water. 

'  Tdpfer's  method,  p.  174. 

*  Determination  of  amino-acid  nitrogen  p.  88. 
'Phenylhydrazine  reaction,  pp.  22  and  413. 

*  Phenylhydrazine  reaction,  pp.  22  and  413. 
'  Determination  of  sugar  in  blood,  p.  279. 

*  Roberts'  ring  test,  pp.  104  and  424. 
^  Rosenheim's  periodide  test,  p.  273. 


6o6  PHYSIOLOGICAL    CHEMISTRY 

Sahli's  Reagent.^ — This  reagent  consists  of  a  mixture  of  equal  parts 
of  a  48  per  cent  solution  of  potassium  iodide  and  an  8  per  cent  solution  of 
potassium  iodate. 

Salted  Plasma. 2 — Allow  arterial  blood  to  run  into  an  equal  volume 
of  a  saturated  solution  of  sodium  sulphate  or  a  10  per  cent  solution  of 
sodium  chloride.  Keep  the  mixture  in  the  cold  room  for  about  24 
hours. 

Schweitzer's  Reagent.^ — Add  potassium  hydroxide  to  a  solution  of 
copper  sulphate  which  contains  some  ammonium  chloride.  Filter  off 
the  precipitate  of  cupric  hydroxide,  wash  it,  and  bring  3  grams  of  the 
moist  cupric  hydroxide  into  solution  in  a  liter  of  20  per  cent  ammonium 
hydroxide. 

Scott-Wilson  Acetone  Reagents.^ — (a)  Mercury  Reagent. — This 
reagent  is  made  up  as  follows:  Mercuric  cyanide  10  grams,  sodium 
hydroxide  180  grams,  water  1200  c.c.  The  solution  is  agitated  in  a  flask 
and  400  c.c.  of  a  0.7268  per  cent  solution  of  silver  nitrate  slowly  run  in. 
Let  stand  three  days  and  decant  supernatant  liquid.  The  silver  nitrate 
solution  is  made  by  taking  i  part  of  standard  silver  nitrate  solution 
(i  c.c.  =  10  mg.  NaCl)  and  3  parts  of  water. 

{h)  Standard  Potassium  Thiocyanate  Solution. — Make  up  an  approxi- 
mately 0.1  per  cent  solution  of  potassium  thiocyanate  and  standardize 
it  against  mercuric  nitrate  or  silver  nitrate.  It  is  convenient  to  have  the 
solution  of  such  strength  that  i  c.c.  =  i  mg.  of  Hg. 

(c)  Acid  Mixture. — Nitric  acid  40  parts,  sulphuric  acid  5  parts,  and 
water  55  parts. 

{d)  Permanganate  N/s  Solution. — Dissolve  6.324  grams  of  potassium 
permanganate  in  water  and  make  up  to  a  liter. 

Seliwanoff's  Reagent.^ — Dissolve  0.05  gram  of  resorcinol  in  100  c.c. 
of  dilute  (i  :  2)  hydrochloric  acid. 

Sherrington's  Solution.*' — This  solution  possesses  the  following 
formula : 

Methylene-blue o .  i  gram. 

Sodium  chloride 1.2  grams. 

Neutral  potassium  oxalate .' 1.2  grams. 

Distilled  water 300 .  o  grams. 

Silver  Nitrate  Solution.^ — Dissolve  29.042  grams  of  silver  nitrate  in 
I  liter  of  distilled  water.     Each  cubic  centimeter  of  this  solution  is 

'  Determination  of  free  acid,  p.  164. 
^  Experiments  on  blood  plasma,  p.  268. 
'  Schweitzer's  solubility  test,  p.  49. 

*  Determination  of  acetone  and  acetoacetic  acid,  pp.  284  and  536. 

*  Seliwanoff's  reaction,  pp.  35  and  447. 

*  "Blood  counting,"  p.  305. 

'  Volhard-Arnold  method,  p.  556,  Mohr's  method,  p.  558,  and  Dehn-Clark  method, 
p.  558. 


REAGENTS   AND   SOLUTIONS  607 

equivalent   to    o.oi    gram   of   sodium   chloride   or   to  0.006   gram   of 
chlorine. 

Sodium  Acetate  Solution.^ — Dissolve  loo  grams  of  sodium  acetate 
in  800  c.c.  of  distilled  water,  add  100  c.c.  of  30  per  cent  acetic  acid 
to  the  solution,  and  make  the  volume  of  the  mixture  up  to  i  liter  with 
distilled  w^atcr. 

Sodium  Alcoholate  (N/io)  Solution.- — The  sodium  alcoholate  is 
made  by  dissolving  2.3  grams  of  cleaned  metallic  sodium  in  i  liter  of 
absolute  alcohol.  It  is  advisable  that  it  be  slightly  weaker  than  stronger 
than  tenth-normal.  It  may  be  standardized  against  pure  benzoic  acid 
in  washed  chloroform.  It  may  also  be  standardized  against  N/io  HCl 
provided  the  alcoholate  solution  contains  not  more  than  traces  of 
carbonate. 

Sodium  Alizarin  Sulphonate.^ — Dissolve  i  gram  of  sodium  alizarin 
sulphonate  in  iDo  c.c.  of  water. 

Sodium  Sulphide  Solution.^ — Saturate  a  i  per  cent  solution  of 
sodium  hydroxide  with  hydrogen  sulphide  gas  and  add  an  equal  volume 
of  I  per  cent  sodium  hydroxide. 

Sodium  Thiosulphate  Standard  (N/io)  Solution.^ — Weigh  out  25 
grams  of  ordinary  c.p.  sodium  thiosulphate  or  24.83  grams  of  the  pure 
dry  recrystallized  salt.  Dissolve  in  water  and  dilute  to  a  liter.  Boiled 
distilled  water  must  be  used.  Keep  in  a  bottle  wdth  a  siphon  arrange- 
ment and  carrying  a  soda  lime  tube  to  exclude  CO2. 

It  is  best  standardized  against  acid  potassium  iodate  KH(I03)2- 
Weight  out  accurately  0.3249  gram  of  acid  potassium  iodate.  Dissolve 
in  50  c.c.  of  water,  heating  gently  if  necessary.  Transfer  the  solution  to 
a  100  c.c.  flask,  rinsing  the  beaker  carefully  and  make  to  mark  with 
water.  This  solution  is  exactly  decinormal.  Pipette  out  25  c.c.  into  an 
Erlenmeyer  flask,  add  i  gram  of  potassium  iodide  dissolved  in  a 
little  water,  and  a  few  cubic  centimeters  of  dilute  hydrochloric  add. 
Titrate  immediately  with  the  thiosulphate  solution.  When  the  solution 
becomes  pale  yellow  add  a  few  cubic  centimeter  of  i  per  cent  solution  of 
soluble  starch  and  titrate  to  loss  of  blue  color. 

Solera's  Test  Paper.  ^' — Saturate  a  good  quality  of  filter  paper  with 
0.5  per  cent  starch  paste  to  which  has  been  added  sufficient  iodic  acid 
to  make  a  i  per  cent  solution  of  iodic  acid  and  allow  the  paper  to  dry 
in  the  air.     Cut  it  in  strips  of  suitable  size  and  preserve  for  use. 

^  Uranium  acetate  method,  p.  552. 

-  Determination  of  hippuric  acid,  p.  519. 

^Topfer's  metliod,  p.  174. 

*  Kruger  and  Schmidt's  method,  p.  513. 

'  Determination  of  acetone  and  acetoacetic  acid,  p.  533. 

*  Solera's  reaction,  p.  59. 


6o8  PHYSIOLOGICAL   CHEMISTRY 

Spiegler's  Reagent.^ — This  reagent  has  the  following  composition: 

Tartaric  acid 20  grams. 

ISIercuric  chloride 40  grams. 

Sodium  chloride 50  grams. 

Glycerol 100  grams. 

Distilled  water 1000  grams. 

Starch  Iodide  Solution.^ — Mix  o.i  gram  of  starch  powder  with 
cold  water  in  a  mortar  and  pour  the  suspended  starch  granules  into  75- 
100  c.c.  of  boiling  water,  stirring  continuously.  Cool  the  starch  paste, 
add  20-25  grams  of  potassium  iodide  and  dilute  the  mixture  to  50  c.c. 
This  solution  deteriorates  upon  standing,  and  therefore  must  be  freshly 
prepared  as  needed. 

Starch  Paste. — Grind  2  grams  of  starch  powder  in  a  mortar  with  a 
small  amount  of  water.  Bring  200  c.c.  of  water  to  the  boiling-point  and 
add  the  starch  mixture  from  the  mortar  with  continuous  stirring.  Bring 
again  to  the  boiling-point  and  allow  it  to  cool.  This  makes  an  approxi- 
mate I  per  cent  starch  paste  which  is  a  very  satisfactory  strength  for 
general  use. 

Stokes'  Reagent.^ — A  solution  containing  2  per  cent  ferrous  sulphate 
and  3  per  cent  tartaric  acid.  When  needed  for  use  a  small  amount 
should  be  placed  in  a  test-tube  and  ammonium  hydroxide  added  until 
the  precipitate  which  forms  on  the  first  addition  of  the  hydroxide  has 
entirely  dissolved.  This  produces  ammonium  ferrotartrate,  which  is  a 
reducing  agent. 

Suspension  of  Manganese  Dioxide.^ — Made  by  heating  a  0.5  per 
cent  solution  of  potassium  permanganate  with  a  little  alcohol  until  it  is 
decolorized. 

Tanret's  Reagent.^ — Dissolve  1.35  grams  of  mercuric  chloride  in 
25  c.c.  of  water,  add  to  this  solution  3.32  grams  of  potassium  iodide 
dissolved  in  25  c.c.  of  water,  then  make  the  total  solution  up  to  60  c.c. 
with  distilled  water  and  add  20  c.c.  of  glacial  acetic  acid  to  the  mixture. 

Tincture  of  Iodine."— Dissolve  70  grams  of  iodine  and  50  grams  of 
postassium  iodide  in  i  liter  of  95  per  cent  alcohol. 

Toison's  Solution.^ — This  solution  has  the  following  formula: 

Methyl  violet o .  025  gram. 

Sodium  chloride i .  o      gram. 

Sodium  sulphate 8.0      grams. 

Glycerol 30.0      grams. 

Distilled  water 160.0      grams. 

'  Spiegler's  ring  test,  pp.  104  and  424. 

*  Fehling's  method,  p.  523. 

'  Hemoglobin,  p.  296.     Hemochromogen,  p.  299. 

*  Kriiger  and  Schmidt's  method,  p.  513. 
'  Tanret's  test,  pp.  104  and  425. 

*  Smith's  test,  pp.  268  and  433. 
'  "Blood  counting,"  p.  305. 


REAGENTS    AND    SOLUTIONS  609 

Topfer's  Reagent  J — Dissolve  0.5  gram  of  di-methylaminoazobenzene 
in  100  c.c.  of  95  per  cent  alcohol. 

Tropaeolin  OO.- — Dissolve  0.05  gram  of  tropaeolin  00  in  100  c.c. 
of  50  per  cent  alcohol. 

Uffelmann's  Reagent. ' — Add  a  5  per  cent  solution  of  ferric  chloride 
to  a  I  per  cent  solution  of  carbolic  acid  until  an  amethyst-blue  color  is 
obtained. 

Uranium  Acetate  Solution.^ — Dissolve  about  35.0  grams  of  uranium 
acetate  in  i  liter  of  water  with  the  aid  of  heat  and  3-4  c.c.  of  glacial 
acetic  acid.  Let  stand  a  few  days  and  filter.  Standardize  against  a 
phosphate  solution  containing  0.005  gram  of  P2O5  per  cubic  centimeter. 
For  this  purpose  dissolve  14.721  grams  of  pure  air-dry  sodium  am- 
monium phosphate  (NaNH4HP04+4H20)  in  water  to  make  a  liter. 
To  20  c.c.  of  this  phosphate  solution  in  a  200  c.c.  beaker  add  30  c.c. 
of  water  and  5  c.c.  of  sodium  acetate  solution  (see  above)  and 
titrate  with  the  uranium  solution  to  the  correct  end  reaction  as  indi- 
cated in  the  method  proper,  page  552.  If  exactly  20  c.c.  of  uranium 
solution  are  required  i  c.c.  of  the  solution  is  equivalent  to  0.005  gram 
P2O5.  If  stronger  than  this  dilute  accordingly  and  check  again  by 
titration. 

Urease.^ — (a)  Soy  Bean  Meal. — Grind  the  soy  bean  to  a  powder 
which  will  pass  through  a  20-mesh  sieve. 

(b)  Solid  Urease  Preparation. — Digest  i  part  of  soy  bean  meal  with 
5  parts  of  water  at  room  temperature,  with  occasional  stirring  for  an 
hour,  and  clear  the  solution  by  filtration  through  paper  pulp  or  centri- 
fugation.  Pour  this  extract  slowly,  with  stirring,  into  at  least  10  volumes 
of  acetone.  The  acetone  dehydrates  the  enzyme  preparation.  Filter, 
dry  in  vacuum  and  powder.  For  standardization  procedure  see  the 
determination  of  urea  in  urine. 

(c)  Enzyme  Solution. — Dissolve  2  grams  of  urease,  prepared  as  above, 
together  with  0.6  gram  of  di-potassium-hydrogen  phosphate  and  0.4 
gram  of  mono-potassium-dihydrogen  phosphate  in  10  c.c.  of  water. 
The  solution  may  be  kept  under  toluol  for  two  weeks,  without  losing 
activity. 

Uric  Acid  Reagents.*"' — (a)  Silver  Magnesium  Solution. — 

3  per  cent  silver  lactate  solution 70  c.c. 

Magnesia  mixture 30  c.c. 

Concentrated  ammonium  hydroxide  solution 100  c.c. 

^  Topfer's  method,  p.  174. 
^Test  for  free  acid,  p.  154. 

*  Uffelmann's  reaction,  p.  170. 

*  Phosphate  determination,  p.  552. 
^  Determination  of  urea,  p.  491. 

*  Determination  of  uric  acid,  p.  274  and  p.  510. 

39 


6lO  PHYSIOLOGICAL    CHEMISTRY 

(b)  Uric  Acid  Reagent  for  Colorimetric  Method. — Place  loo  grams  of 
sodium  tungstate,  80  c.c.  of  85  per  cent  phosphoric  acid,  and  750  c.c.  of 
distilled  water  in  a  liter  flask.  Boil  the  mixture  with  a  reflux  condenser 
for  two  hours,  cool  and  dilute  to  i  liter,  filtering  if  necessary. 

(c)  Sodium  Carbonate  Solution. — Dissolve  200  grams  anhydrous 
sodium  carbonate  in  warm  water  and  make  up  to  i  liter. 

{d)  Uric  Acid  Formaldehyde  Standard. — Place  i  gram  of  uric  acid  in 
a  liter  volumetric  flask  and  dissolve  with  200  c.c.  of  a  0.4  per  cent  lithium 
carbonate  solution.  Add  to  the  solution  40  c.c.  of  40  per  cent  formalde- 
hyde solution,  shake  the  mixture  and  allow  to  stand  for  a  few  minutes. 
Acidify  the  clear  solution  with  20  c.c.  of  normal  acetic  acid  and  dilute 
to  mark  with  distilled  water  The  solution  should  remain  perfectly 
clear  and  the  next  day  (not  before)  may  be  standardized  against  a 
freshly  prepared  solution  of  uric  acid  in  lithium  carbonate.  The  color 
produced  by  5  c.c.  of  this  solution  corresponds  very  closely  to  that 
produced  from  i  mg.  of  pure  uric  acid.  The  colorimeter  reading  ob- 
tained from  the  solution  when  thus  compared  with  pure  uric  acid  (i  mg.) 
is,  of  course,  thereafter  used  as  the  standard  value  corresponding  to  i  mg. 
of  uric  acid. 

{e)  Uric  Acid  Standard  in  Phosphate  Solution. — Dissolve  9  grams  of 
pure  crystallized  disodium  hydrogen  phosphate,  together  with  i  gram 
of  crystallized  sodium  dihydrogen  phosphate,  in  200  to  300  c.c.  of  hot 
water,  and  filter  if  the  solution  is  not  perfectly  clear.  Make  this 
filtrate  up  to  about  500  c.c.  with  hot  water,  and  pour  this  hot  or  warm 
(and  perfectly  clear)  solution  upon  exactly  200  mg.  of  pure  uric  acid 
suspended  in  a  few  cubic  centimeters  of  water  in  a  liter  volumetric 
flask.  Agitate  the  mixture  for  a  few  minutes  until  the  uric  acid  com- 
pletely dissolves.  Cool,  add  exactly  1.4  c.c.  of  glacial  acetic  acid, 
dilute  to  the  mark,  and  mix.  Add  about  5  c.c.  of  chloroform  to  pre- 
vent the  growth  of  bacteria  or  moulds  in  the  solution.  Five  c.c.  of 
this  solution  contain  exactly  i  mg.  of  uric  acid. 

The  solution  of  uric  acid  in  the  phosphate  solution  is  very  readily 
prepared,  does  not  need  to  be  standardized,  and  appears  to  keep 
indefinitely. 


REAGENTS    AND    SOLUTIONS 


6ll 


INTERNATIONAL  ATOMIC  WEIGHTS,  191 6 


0=  16. 

Aluminium Al         27.1 

Antimony Sb  120.2 

Arsenic As         74  96 

Barium Ba  137-37 

Bismuth Hi  208.0 

Boron B  11 .0 

Bromine Br         79.92 

Cadmium Cd  1 1 2  .  40 

Calcium Ca        40.07 

Carbon C  12. 005 

Chlorine CI         35  46 

Chromium Cr         52.0 

Cobalt Co  '58.97 

Copper Cu        63.57 

Fluorine F  19.0 

Glucinum Gl  9.1 

Gold Au  197.  2 

Hydrogen H  i .  008 

Iodine I  126.92 

Iridium Ir  193 .  i 

Iron Fe         55  84 

Lanthanum La  1390 

Lead Pb  207 .  20 

Lithium Li  6.94 

Magnesium ^Ig       24.32 


0=  16. 

Manganese Mn  54. 93 

Mercury Hg  200.6 

Molybdenum Mo  96.0 

Nickel Ni  58.68 

Nitrogen N  14.01 

Osmium Os  190.9 

Oxygen O  16.00 

Palladium Pd  106 .  7 

Phosphorus P  31   04 

Platinum Pt  195 .  2 

Potassium K  39- 10 

Radium Ra  226.0 

Selenium Se  79  •  2 

Silicon Si  28.3 

Silver Ag  107.88 

Sodium Na  23 .  00 

Strontium Sr  87 .  63 

Sulphur S  32.06 

Tantalum Ta  181 .  5 

Tellurium Te  127.5 

Tin Sn  118.  7 

Titanium Ti  48.  i 

Tungsten W  184.0 

Ui'anium U  238.  2 

Zinc Zn  65.37 


INDEX 


Main  references  are  in  heavy-faced  type. 


Abderhalden  test  for  pregnancy,  principle  of,  3 
Absorption  of  carbohydrate  as  influenced  by  fat 

ingestion,  568 
Acacia  solution,  formation  of  emulsion  by,  178 
Acetoacetic  acid,  270,  284,  412,  438,  539 
Amold-Lipliawsky  test  for,  440 
formula  for,  438 
Gerhardt's  test  for,  439 
Hurtley's  reaction  for,  440 
Le  Nobel  reaction  for,  439 
quantitative  determination  of,  284,  294,  S39 
Acetone,  270,  284,  294,  412,  43S,  538 
bodies,  270,  284,  435i  533 

determination  of  in  blood,  284 
of  in  urine,  533 
formula  for,  435 
Frommer's  test  for,  438 
Gunning's  iodoform  test  for,  436 
Legal's  iodoform  test  for,  437 
Lieben's  test  for,  437 

quantitative  determination  of,  284,  294,  538 
Reynolds-Gunning  test  for,  438 
Rothera's  reaction  for,  438 
Acholic  stool,  223 
Achroo-dextrins,  43,  56 
a-achroo-dextrin,  56 
0-achroo-dextrin,  56 
-)-achroo-dextrin,  56 
Acid,  acetic,  335,  370,  397 

acetoacetic,  270,  284,  412,  438,  539 

alloxyproteic,  369,  392 

amino-acetic,  69,  71 

amino-butyric,  214 

amino-ethyl-sulphonic,  204,  346 

a-amino-/3-hydroxy-propionic,  69,  73 

a-amino-^-imidazol-propionic,  68,  77 

a-amino-iso-butyl-acetic,  69,  79 

a-amino-/S-methyl-^-ethyl-propionic,  68,  80 

a-amino-normal  glutaric,  69,  82 

ot-amino-propionic,  68,  72 

amino-succinic,  69,  82 

amino- valerianic,  214 

a-amino-iso- valerianic  (see  Valine),  69,  78 

a-diamino-^-dithiolactyl,  69,  75 

aspartic,  69,  82 

benzoic,  72,  369.  390,  395,  583.  S86 

butyric,  8,  319,  323,  370,  397 

caproic,  313,  319 

carbamic,  247 

cholic,  204 

chondroitin-sulphuric,  335,  369,  392 

citric,  313 

combined  hydrochloric  (protein  salt),  56,  I40j 

I7S 
cyanuric,  373 
a-«-diamino-caproic,  68,  80 


Acid,  diaminotrihydroxydodecanoic,  68,  85 
diazo-benzene-sulphonic,  454 
ethereal  sulphuric,  212,  369,  385 
fatty,  176,  178.  182,  212.  370,  397 
formic,  26,  370,  397 

free  hydrochloric,  56,  140,  155,  164,  175 
glucothionic,  467 
glutamic,  69,  82 

glycerophosphoric,  353.  354.  370,  399 
glycocholic,  204 
glycosuric,  395 
glycuronic,  36,  38,  442 
glyoxylic,  98 

guanidine-or-amino-valerianic,  68,  79 
hippuric,  72,  369.  388,  390,  396,  585 
homogentisic,  26,  369,  394,  417 
hydroxymandelic,  369.  395 
iminazolpropionic,  214 
indolacetic,  214.  452 
indole-ot-amino-propionic,  68,  77 
indoxyl-sulphuric,  212,  369,  385 
inosinic,  341,  346 
kynurenic,  369,  394 
lactic.  40,  140,  159.  170,  3i4i  340,  342 
lauric,  313 

mucic,  36,  40,  445.  446 
myristic,  313 

nucleic   94.  123.  I24.  I30-I33 
osmic,  356 
oxalic,  369.  391.  545 
oxaluric,  369,  397 

oxy-a-pyrrolidine-carboxylic,  68,  8s 
oxyproteic,  369,  392 

palmitic,  176.  i77,  181,  182 

para-cresolesulphuric,  369,  385 

para-oxyphenyl-acetic,  212,  214,  369,  394 

para-oxy-^-phenyl-o-amino-propionic,  69,  74 
86 

para-oxyphenyl-propionic.  212,  214,369.394 

paralactic,  247,  314.  342.  370,  398 

phenaccturic,  370.  398 

phenol-sulphuric,  369,  38s 

phenylacetic,  214 

phcnyl-a-amino  propionic,  69.  73 

phenylpropionic,  214 

phosphocarnic,  341,  346,  370.  399 

phosphoric.  406 

pyrocatechin-sulphuric,  369,  385 

a-pyrrolidine-carboxylic,  68.  85 

sarcolactic,  342 

skatole  acetic,  77 

skatole  carbonic,  217,  220 
,  skatoxyl-sulphuric,  369,  385 

stearic.  177,  354 

succinic,  214 

sulphanilic,  455 
613 


6i4 


INDEX 


Acid,  tannic,  45,  48,  103 
taurocholic,  204 
trichlorethylglucuronic,  417 
uric.  26,  127,  247,  274.  341.  369.  371,  377.  438, 

460,  475.  510 
urocanic,  370,  398 
uroferric,  369,  392 
uroleucic,  369 

volatile  fatty,  212,  215,  370,  397 
Acid  albuminate.     See  Acid  metaprotein. 
Acid-forming  foods,  influence  of  on  hydrogen  ion 

concentration  of  urine,  580 
Acid  infraprotein.     See  Acid  metaprotein. 
Acid  metaprotein,  95,  115,  116 
coagulation  of,  116 
experiments  <-  i.  116 
precipitati'    .    ■;,  ii6 
preparation  of,  116 
solubility  of,  116 
sulphur  content  of,  116 
Acidity  of  gastric  juice,  quantitative  determina- 
tion of,  148,  IS9,  162 
urine,  cause  of,  361,  406 

quantitative   determination    of,   by   hy- 
drogen ion  concentration,  840 
by  titration,  479 
Acidosis,  441,  576 

metabolism  in,  576 
Acid-hematin,  254,  269,  299 
Acree-Rosenheim  formaldehyde  reaction,  100 
Acrolein,  formation  of,  from  olive  oil,  180 

from  glycerol,  183 
Activation,  6,  187 
Activation  by  calcium  salts,  187 
Adam's  paper  coil  method  for  determination  of 

fat  in  milk,  326 
Adaptation,  57 
Adenase,  4 

Adenine,  4,  125,  126,  127,  132,  347,  370,  401 
Adipocere,  179 

Adrenaline,  determination  of  in  adrenals,  290 
Agar-agaj-,  20,  50,  22J,  589 
Agglutination,  250,  262 
Alanine,  68,  72,  214 
Albumin,  egg,  93,  107 

crystallized,  preparation  of,  106 
powdered,  preparation  of,  107 

tests  on,  107 
serum,  93,  245,  268,  412,  422 
solution,  preparation  of,  97 
Albumin  in  urine,  412,  422 

acetic  acid  and  potassium  ferrocyanide 
test  for,  425 
coagulation  or  boiling  test  for,  424 
determination  of,  531 
Heller's  ring  test  for,  /<23 
Roberts'  ring  test  for,  424 
sodium  chloride  and  acetic  acid  test  for,  425 
Spiegler's   ring   test   for,    42J 
Tanret's  test  for,  425 
tests  for,  423 
Albumins,  93,  95,  96 
Albuminates.     See  Metaproteins. 
Albuminates,  formation  of,  by  metallic  salts,  102 
Albuminoids,  93,  112,  330 
Albumoscope,  103,  423 
Albumoses  (see  Proteoses,  p.  118) 
Alcohol-soluble  proteins  (see  Prolamins,  p.  1 1 1) 


Alcoholic-zinc  chloride  test  for  urobilin,  401 

Aldehyde,  25,  42 

Aldehyde  group,  39 

Aldehyde  test  for  alcohol,  42 

v.  Aider's  method  of  detecting  proteose  in  urine, 

427 
Aldose,  19 

Aliphatic  nucleus,  65,  68 

Alizarin  yellow  R,  use  of,  as  indicator,  157,  158 
Alkali  albuminate.     See  Alkali  metaprotein. 
Alkali-hematin,  254,  298 
Alkali  metaprotein,  95,  115,  116 
experiments  on,  116 
precipitation  of,  116 
preparation  of,  116 
sulphur  content  of,  116 
Alkaline  tide,  362,  577 

demonstration  of,  577 
AUantoin,  369,  392 

crystalline  form  of,  392 
experiments  on,  393 
formula  for,  392 

preparation  of,  from  uric  acid,  393 
quantitative  determination  of,  by  Wiechow- 
ski-Handovsky  method,  518 
by  difference,  510 
separation  of,  from  urine,  393 
Allen's  modification  of  Fehling's  test,  419 
Alm^n's  reagent,  preparation  of,  429 
Alloxyproteic  acid,  369,  392 
Aloin-turpentine  test  for  "occult  blood,"  73s 
Aluminium    hydroxide,     use    of,     in    removal   of 

protein,  276,  329 
Amalgamation  test  for  mercury,  450 
Amandin,  93 
Amide  nitrogen,  64 

Amino  acids,  65,  67.  68,   186,  195,  248,  270,  369, 
394 
group,  65,  88,  99 

preparation  of,  in  crystalline  form,  &S,  87 
a-amino-^-hydroxy-propionic  acid,  69,  73 
a-amino-/3-imidazol-propionic  acid,  68,  77 
a-amino-iso-butyl-acetic  acid,  69,  78 
«-amino-normal-glutaric  acid,  69,  82 
Amino-butyric  acid,  214 
Amino-nitrogen,    quantitative    determination    of, 

88,  91,  277,  502 
Amino-succinic  acid,  69,  82 
Amino-valerianic  acid,  214 
cr-amino-iso-valerianic  acid,  69,  78 
Ammonia,  64,  67,  71,  107 
in  blood,  247,  270 

quantitative  determination  of,  278 
in  urine,  370,  402,  soi,  S7S 

quantitative  determination  of,  499 
Ammoniacal  cupric  hydroxide,  solubility  test,  49 
Ammoniacal  silver  solution,  preparation  of,  594 
Ammoniacal-zinc  chloride  test  for  urobilin,  400 
Ammonium     benzoate,     synthesis    of,     to    form 

hippuric  acid,  585 
Ammonium      magnesium      phosphate      ("Triple 
phosphate"),  363,  408 
in  urinary  sediments,  458 
Ammonium  purpurate,  381 
Ammonium  urate,  363,  377,  461,  sn 

crystalline  form   of,    Plate   VI,   opposite 
p.  462 
Amphopeptone,  95,  ii9   , 


INDEX 


6l 


Amygdalin,  4 

Amylase,  pancreatic,  4,  11,  187,  192 

digestion  of  dry  starch  by,  188,  192 

inulin  by,  192 
experiments  on,  11,  191 
influence  of  bile  upon  action  of,  192 
most  favorable  temperature  for  action 
of,  192 
salivary,  4,  10.  55,  140 

activity  of,  in  stomach,  57,  140 
experiments  on,  10,  59 
inhibition  of  activity  of,  57,  61 
nature  of  action  of,  56 
products  of  action  of,  56 
vegetable,  4,  10 
Amylases,  4,  10,  SS.  187 

experiments  on,  10,  59,  191,  239 
Amyloid,  49,  113 
Amylolytic  enzymes.     See  Amylases. 

quantitative    determination   of    activity 
of,  192,  239 
Anabolism,  564 
Animal  parasites  in  feces,  227 

in  urinary  sediments,  465,  474 
Anti-enzymes,  9 

experiments  on,  17 
Antimony  pentachloride  as  cellulose  solvent.    50 
Antimony  trichloride  as  cellulose  solvent,  49 
Antipepsin,  9,  17 
Antipeptone,  95,  119 
Antirennin,  9 
Antithrombin,  257 
Antitrypsin.  9,  i8 
Aporrhegmas,  214 
Arabinose.  19,  37,  443 

Bial's  reaction  for,  37,  443 
orcinol  test  on,  38.  444 
phenylhydrazine  test  on,  38 
Tollens'  reaction  on,  38,  443 
Arginase,  4 

Arginine,  4.  67,  68,  79,  i86 

Arnold-Lipliawsky  reaction  for  acetoacetic  acid, 
440 
reagent,  preparation  of,  440 
Aromatic  oxyacids,  369,  394 
Arsenic  in  urine,  detection  of,  448 

determination  of,  448 
Ascaris,  17,  18 

Ash  of  milk,  quantitative  determination  of,  327 
Asparagine,  82 

formula  for,  82 
Aspartic  acid,  65,  67,  69,  82 

crystalline  form  of,  81 
formula  for,  82 
Assimilation  limit,  2t 

Assimilation  limit  of  dextrose,  21,  413.  568 
Atkinson  and  Kendall's  hemin  test,  266 
Autolytic  enzymes,  3 
Azolitmin,  use  of,  as  indicator,  158 

Babcock  fat  method,  324 

tube,  324 
Bacteria  iiT  feces.  221.  224,  225,  588 

quantitative  determination  of.  241 
Bacterial  nitrogen  in  feces,  226,  588 
determination  of,  241 
Balance,  metabolic  preparation  of,  592 
calcium,  592 


Balance,  magnesium,  592 
nitrogen,  592 
phosphorus,  592 
sulphur,  592 
Bang  reduction  flask,  281 
Bang's  method  for  estimation  of  sugar  in  blood. 

280 
Bang's  method  for  estimation  of  sugar  in  urine,  524 
Bardach's  reaction,  loi 

Barfoed's  reagent,  preparation  of,  30.  200,  595 
Barfoed's  test  for  monosaccharides,  30 
Baryta  mixture,  preparation  of,  37s,  S9S 
Base-forming  foods,  influence  of  on  hydrogen  ion 

concentration  of  urine,  362,  580 
Basic  lead  acetate  solution,  542,  596 
Bayberry  tallow,  saponification  of,  181 

source  of,  181 
Bayberry  wax.     See  Bayberry  tallow,  i8i 
Bead  test  (Einhorn),  238 
Beckmann-Heidenhain  apparatus,  365 
"  Bence-Jones'  protein,"  detection  of,  428 
Benedict  creatine  preparation,  384 
Benedict    and    Bock's   method    for   quantitative 

determination  of  total  nitrogen,  488 
Benedict  and  Lewis  method  for  sugar  in  blood,  279 
Benedict-Folin  creatinine  preparation,  383 
Benedict-Folin  method  for  creatine  in  urine,  508 
Benedict-Hitchcock  method  for  uric  acid  in  urine, 

Sio 
Benedict- Murlin  method  for  amino-acid  nitrogen 

in  urine,  504 
Benedict-Pearce  method  for  sugar  in  blood,  280 
Benedict's      method      for      quantitative      deter- 
mination of  sugar,  522 
Benedict's      method      for      quantitative      deter- 
mination of  sulphur,  548 
Benedict's      method      for      quantitative      deter- 
mination of  urea,  494 
Benedict's  method  for  uric  acid  in  blood,  275 
Benedict's  modifications  of  Fehling's  test.  27,  417 
solution,     for     use     in     quantitative     deter- 
mination of  sugar,  preparation  of,  522.  596 
solutions,  preparation  of,  27,  417,  596 
sulphur  reagent,  preparation  of.  597 
Benzidine    peroxidase    reaction    (Wilkinson    and 

Peters).  321 
Benzidine  reaction  for  blood,  171,  233,  263,  431 
Benzoic  acid.  72,  369,  390,  39s,  583.  585 
crystalline  form  of.  396 
experiments  upon,  396 
formula  for,  395 
solubility  of,  396 
sublimation  of,  396 
Bergeim's     modification    of     the     Herter-Foster 

method  for  indole  in  feces.  242 
Bergeim's  phosphonuclease  theory  for  the  origin 

of  hydrochloric  acid  of  gastric  juice,  141 
Berthelot-Atwater  bomb  calorimeter.  586 
Bertrand's  method  for  sugar  determination,  527 
Bial's  reaction  for  pentoses,  37.  443 
Bial's  reagent,  preparation  of,  37.  443 
Bile,  171.  203,  412.  432 
analysis  of.  203. 
constituents  of,  203 
daily  secretion  of.  203 
freezing-point  of,  203 
influence  on  digestion,  gastric.  147 
pancreatic,  178,  190 


6i6 


INDEX 


Bile,  inorganic  constituents  of,  203,  209 
nucleoprotein  of,  203,  207 
reaction  of,  202  207 
secretion  of,  202 
specific  gravity  of,  203, 
Bile  acids,  204 

Hay's  test  for,  209 
Mylius's  test  for,  208 
Neukomm's  test  for,  209 
Oliver's  test  for,  209 
Pettenkofer's  test  for,  208 
tests  for,  208 

V.  Udr4nsky's  test  for,  208 
Bile  acids  in  feces,  detection  of,  236 
Bile  acids  in  urine,  412,  434 

Hay's  test  for,  434 
Myliys's  test  for,  434 
Neukomm's  test  for,  43s 
Oliver's  test  for,  43s 
Pettenkofer's  test  for,  434 
tests  for,  434 

V.  Udrdnsky's  test  for,  434 
Bile  pigments,  203,  204,  207 

Gmelin's  test  for,  207 
Hammarsten's  reaction  for,  207 
Huppert's  reaction  for,  207 
Xakayama's  reaction  for,  207 
Rosenbach's  test  for,  207 
Smith's  test  for,  208 
tests  for,  207 
Bile  pigments  in  urine,  412,  432 

Gmelin's  test  for,  433 
Hammarsten's  reaction  for,  .133 
Huppert's  reaction  for,  433 
Nakayama's  reaction  for,  433 
Rosenbach's  test  for,  433 
Salkowski-Schipper's    reaction    for, 

433 
Salkowski's  test  for,  433 
Smith's  test  for,  433 
tests  for,  432 
Bile  salts,  204,  434 

crystallization  of,  204,  209 
Biliary  calculi,  206,  209 
analysis  of,  209 
Bilicyanin,  204 
Bilifuscin,  204 
Bilihumin,  204 
Biliprasin,  204 
Bilirubin,  204 

crystalline  form  of,  205 
in  urinary  sediments,  458,  464 
BJliverdin,  204,  206 
"Biological"  blood  test,  258 
Bismuth,  influence  on  color  of  feces,  222,  238 
Bismuth  reduction  tests,  29,  420 
Bismuth  test  for  choline,  358 
Biuret,  99.  373,  375 

formation  of,  from  urea,  99,  373 
Biuret  paper  of  Kantor  and  Gies,  100 
Biuret  potassium  cupric  hydroxide.     See  Cupri- 
potassium  biuret,  99 
test,  98 

Posner's  modification  of,  100 
Biuret  reagent  (Gies),  preparation  of,  100 
Black's  method  for  determination  of  0-hydroxy- 
butyric  acid,  541 
reaction  for  ^-hydroxybutyric  acid,  441 


Black's  reagent,  preparation  of,  441 
Blood,  171,  225,  233,  245,  270,  412,  429 
acetoacetic  acid  in,  270    284 
acetone  in,  247,  270,  284 
agglutination  of,  250,  262 
amino-acid  nitrogen  in,  270 
amino-acids  in,  247,  248 
ammonia  in,  270,  278 
Bordet  test  for,  258 
Blood,  cholesterol  in,  247,  270,  285 
coagulation  of,  257 

Howell's  theory  of,  257 
composition  of  normal  and  pathological,  270 
constituents  of,  247,  248 
creatine  in,  248,  270,  276 
creatinine  in,  248,  270,  276 
crystallization  of  oxyhemoglobin  of,  250,  266 
defibrinated,  257,  260 
detection  of,  171,  233,  263,  429 
determination  of  acetone  in,  284,  294 
acetoacetic  acid  in,  284,  294 
amino  acid  nitrogen  in,  277 
ammonia  in,  278 
chlorides  in,  286 
cholesterol  in,  285 
creatine  in,  276 
creatinine  in,  276 
fat  in,  295 

/3-hydroxybutyric  acid  in,  284,  294 
hydrogen  ion  concentration  in,  288 
non-protein  nitrogen  in,  271 
sugar  in,  279 
total  nitrogen  in,  279 
total  solids  in,  288 
urea  in,  274 
uric  acid  in,  274 
erythrocytes  of,  249,  261,  472 
experiments  on,  260,  270 
fat  in,  247,  295 
form  elements  of,  249 
guaiac  test  for,  23s,  258,  262,  430 
hemin  test  for,  264,  429 
fl-hydroxybutyric  acid  in,  270,  284 
in  arthritis,  270,  271 
in  cholelithiasis,  247,  270,  271 
in  diabetes,  270,  271 
in  gout,  270,  271 
in  lipemia,  270,  271 
in  nephritis,  270,  271 
in  uremia,  270,  271 
in  urine,  412.  429 
leucocytes  of,  255 
medico-legal  tests  for,  257,  203 
menstrual,  257 
microscopical  examination  of.  249,  260,  261 

269 
non-protein  nitrogen  of,  270 

determination  of,  271 
nucleoprotein  of,  246,  247 
"occult,"  in  feces,  22s,  233 
ortho-tolidin  test  for,  171,  233,  263,  430 
oxyhemoglobin  of,  250.  296 
pigment  of,  250 
plaques,  256 
plasma,  245,  268 
platelets,  256 
plates,  256 
preparation  of  hematin  from,  266 


INDEX 


617 


Blood  preparation  of  "laky,"  24s,  261 
reaction  of,  24s,  260 
serum,  247,  267 
specific  gravity  of,  24s,  260 
spectroscopic  examination  of,  296 
sugar  in,  247,  270,  271 
test  for  iron  in,  261 
total  amount  of,  24s 

V.  Zeynek  and  Nencki's  hemin  test  for,  266 
Blood  analysis,  270 
Blood  casts  in  urine,  465,  469 
Blood  corpuscles,  249,  255,  261,  472 

"counting,"  304,  308 
Blood  dust,  245,  256 
Blood  in  urine,  412,  429 

benzidine  reaction  for,  431 

guaiac  test  for,  430 

Teichmann's  hemin  test  for,  429 

Heller's  test  for,  429 

Heller- Teichmann  reaction  for,  430 

ortho-tolidin  test  for,  430 

Schumm's   modification   of   guaiac   test 

for,  430 
spectroscopic  examination  of,  431 
tests  for,  429 

V.  Zeynek  and  Nencki's  hemin  test  for, 
430 
Blood  plasma,  245,  268 

constituents  of,  24s 
effect  of  calcium  on  oxalated,  268 
experiments  on,  268 
preparation  of  fibrinogen  from,  268 
oxalated,  268 
salted,  268 
Blood  serum,  247,  267 

coagulation  temperature  of,  267 
constituents  of,  247,  267 
experiments  on,  267 
precipitation  of  proteins  of,  267 
separation  of  albumin  and  globulin  of,  268 
sodium  chloride  in,  268 
sugar  in,  268 
Blood  stains,  examination  of,  269 
Bloor's  nephelometer,  cut  of,  291 
Boas'  reagent,  as  indicator,  155 

preparation  of,  155  * 

Bock  and  Benedict's  apparatus,  cut  of,  489 
Bock  and   Benedict's  microchemical  method  for 

total  nitrogen  in  urine,  488 
Boettger's  test  for  sugar,  29,  420 
"Bolting"  of  food,  influence  of,  on  food  residues 

in  feces,  590 
Bomb  calorimeter,  Berthelot-Atwatcr,  586 
Bonanno's  reaction,  208,  434 
Bonanno's  reagent,  preparation  of,  208,  434 
Bone,  constituents  of,  336-338 
ossein  of,  preparation  of,  336 
quantitative  composition  of,  236,  237 
Bone  ash,  scheme  for  analysis  of,  338 
Borchardt's  reaction  for  fructose,  35,  447 
Bordet  test,  detection  of  human  blood  by,  258 
Boric  acid  and  borates  in  milk,  detection  of,  324 
Bottu's  reagent,  preparation  of,  23,  415 
Bottu's  test,  23,  41  s 
Bromelin,  s 

Bromine  test  for  melanin,  452 
for  tryptophane,  189 
Buccal  glands,  54 


Buffy  coat,  formation  of,  247 
Bunge's  mass  action  theory,  140 
Burker's  hemocytometer,  308 
Butter,  composition  of,  319 
Butyric  acid,  8,  319.  321,  370,  397 
Butyrin,  177.  3i3 
Bynin,  93,  11 1 

Cadaverin,  81,  212 
Calcium  in  urine,  370,  409 

quantitative  determination  of,  559 

balance,  preparation  of,  592 

carbonate  in  urinary  sediments,  458,  459 

caseinate,  318 

in  bone,  detection  of,  337-8 

in  feces,  estimation  of,  592 

oxalate,  391,  458 

in  urinary  sediments,  458 
paracasein,  143,  315 

phosphate  in  urinary  sediments,  458,  460 
in  bone,  detection  of,  337-8 
in  milk,  313,  322 
sulphate  in  urinary  sediments,  458,  460 
Calculi,  biliary,  206,  209 
urinary,  475 

calcium  carbonate  in,  476 
oxalate  in,  476 
cholesterol  in,  478 
cystine  in,  476 
fibrin  in,  478 
indigo  in,  478 
phosphates  in,  476 
uric  acid  and  urates  in,  476 
urostealiths  in,  479 
xanthine  in,  476 
Calliphora,    larvae    of,    formation    of    fat    .from 

protein  by,  179 
Cambogia,  influence  of,  on  color  of  feces,  238 
Calomel,  influence  on  color  of  stool,  222,  238 
Camphor  as  urine  preservative,  368 
Cane  sugar  (see  Sucrose,  p.  41) 
Canton  silk.  69 
Caproic  acid,  313,  319 
Carbamic  acid,  247 
Carbocyclic  nucleus,  65.  68-9 
Carbohydrosis,  4 
Carbohydrates,  19 

absorption  of,  as  influenced  by  fat  ingestion, 

S68 
classification  of,  19 
composition  of,  19 
control  of  putrefaction  by,  213 
in  feces,  estimation  of,  591 
protein-sparing,  action  of,  S79 
review  of,  51 

scheme  for  detection  of.  SJ 
variation  in  solubility  of,  20 
Carbonates  in  urine,  370,  410 
Carbon  moiety  of  protein  molecule,  179 
Carbon  monoxide,  hemoglobin,  254,  255,  297 
potassium  iodide,  test  for,  298 
tannin  test  for,  298 
Carboxylase,  4,  10,  31,  421 
Carmine,  use  in  feces  separation,  239,  587 
Carmine-fibrin,  preparation  of,  12 
Carnine,  341 
Carnitine,  341 

formula  for,  346 


6i8 


INDEX 


Carnomuscarine,  341 
Carnosine,  341,  346 
Carotin,  319 
Cartilage,  335 

constituents  of,  33s 

experiments  on,  335 
Hopkins-Cole  reaction  on,  33s 
Millon's  reaction  on,  335 

preparation  of  gelatin  from,  335 

solubility  of,  335 

unoxidized  sulphur  in,  335 

xanthoproteic  test  on,  335  1 

Casein,  67,  i43,  3i3.  3iS.  318 

decomposition  of,  67 

quantitative  determination  of,  327-329 

soluble,  128,  236 

action  of  rennin  upon,  143,  313 

biuret  test  on,  322 

Millon's  test  on,  322 

precipitation  of,  321 

preparation  of,  321 

quantitative  determination  of.  Hart's  method 
for,  327 

solubility  of,  322 

test  for  phosphorus  in,  322 

test  for  unoxidized  sulphur  in,  322 
Caseinate,  calcium,  318 
Caseinogen.      See  Casein. 
Casts,  46s,  467 

blood,  46s,  469 

epithelial,  465,  469 

fatty,  46s.  469 

granular,  465,  468 

hyaline,  .-165,  468 

pus.  46s,  472 

waxy,  465,  470 
Casts  in  urinary  sediments,  465,  467 
Catabolism,  564 
Cat  gut,  146 
Catalase,  5,  15 

animal,  16 

experiments  on,  16 

quantitative  determination  of,  16 

vegetable.  16 
Catalysis,  2 
Cell.  S65 
Cellulose,  20,  48 

action  of  Schweitzer's  reagent  on,  49 

hydrolysis  of,  49 

iodine  test  on,  49 

solubility  of,  49 

solvents,  49 

utilization  by  animals,  48 
Cellulose  group,  20 
Cerebrin  (cerebroside,)  353.  355.  357 

experiments  on,  357 

hydrolysis  of,  357 

microscopical  examination  of,  357 

preparation  of,  337 

solubility  of,  357 
Cerebro-spinal  fluid,  choline  in,  354 
Cerebrosides,  353.  3SS 
Charcot-Leyden  crystals,  225 

form  of,  225 
Chlorides  in  blood,  270,  286 

in  urine,  370,  405,  556 
detection  of,  406 
quantitative  determination  of,  536 


Cholecyanin,  206 
Choleprasin,  204 

Cholera-red  reaction  for  indole,  218 
Cholesterol,  203,  210,  247,  270,  271,  285,  313,  353, 
355 
crystalline  form  of,  210 
formula  for,  355 
in  blood,  247,  270,  271,  285 

determination  of,  285 
iodine-sulphuric  acid  test  for,  210,  357 
isolation  of,  from  biliary  calculi,  209 
Liebermann-Burchard  test  for,  210,  357 
occurrence  of,  in  urinary  sediments,  458,  463 
origin  of,  356 

preparation  of,  from  nervous  tissue,  356 
Salkowski's  test  for,  210,  357 
Schiff's  reaction  for,  210,  357 
tests  for,  210,  357 
Choletelin,  204 
Choline,  212,  354,  357 

as  putrefaction  product,  212 
formula  for,  354 
tests  for,  357 
Chondrigen,  113 
Chondroalbumoid,  335 
Chondromucoid,  113,  335 
Chondroitin,  335 

Chondroitin-sulphuric  acid,  335,  369,  392 
Chondrosin,  33s 

reducing  action  of,  335 
Chromoproteins  (see  Hemoglobins),  113 
Chyle,  260 

Cipollina's  test,  23,  J14 
Clark's     modification     of     Dehn's     method     for 

determination  of  chlorides,  558 
Cleavage    products    of    protein    (see    Decompo- 
sition products),  64,  68-69 
Clupeine,  67,  94 
Coagulases,  4 

Coagulated  proteins,  95,  116 
biuret  test  on,  118 
digestion  of,  118 
formation  of,  1 16 
Hopkins-Cole  reaction  on,  118 
Millon's  reaction  on,  118 
solubility  of,  118 
xanthoproteic  reaction  on,  118 
Coagulation  of  blood,  256 

Howell's  theory  of,  257 
Coagulation  of  proteins,  116 

changes  in  composition  during,  117 
fractional,  105,  117 
Coagulation  temperature  of  proteins,  105,  117  « 
apparatus  used  in  determining,  106 
method  employed  in  determining,  105 
Co-enzyme,  7 
Cochineal,  use  of,  158 
Collagen,  93.  112,  331.  332 
experiments  on,  332 
percentage  of,  in  ligament.  334 

in  tendon,  331 
production  of  gelatin  from,  333 
solubility  of,  333 
transformation  of,  332,  333 
Collection   and    preservation   of    feces    in    meta- 
bolism experiments,  588 
of  urine  in  metabolism  experiments,  565 
Collodion  dialyzer,  24 


INDEX 


619 


Colloidal  solution,  313 
Colloids,  313,  340 

tissue,  340 
Colostrum,  315.  316,  319 

microscopical  appearance  of,  315 
Combined   hydrochloric   acid   (protein  salt),   56 
140,  175 
preparation  of.  599 
tests  for,  175 
Combustion  of  foodstuffs,  584 
Composition  of  common  foods  (Table),  569 
Compound  test  for  lactose  in  urine,  44s 
Congealing-point  of  fat,  184 
Congo  red,  as  indicator,  153,  154 

preparation  of,  599 
Congo-red  fibrin,  preparation  of,  12 
Conjugated  proteins,  9J,  112 
classes  of,  94,  112 
nomenclature  of,  94,  112 
occurrence  of,  9J,  112 
Conjugate  glycuronates,  26,  J 17,  442 

naphthoresorcinol  reaction  for,  442 
polariscopic-fermentation  test  for,  442 
reduction-polariscopic  test  for,  443 
Connective  tissue,  330 
Constipation,  aid  in,  51,  223 
Cowie's  guaiac  test,  235 
Cramer's  mercuric  oxide  test  for  reducing  sugar, 

28,  419 
Creatine,  247,  248.  270,  276,  341.  342,  350,  369. 
384,  412 
crystalline  form  of,  342 
diacetyl  reaction  for,  350 
formula  for,  346 

quantitative  determination  of,  276,  508 
separation  of,  from  meat  extract,  350 
transformation  into  creatinine,  350 
Creatinine,  26,  248.  270,  369,  381 
coefficient,  definition  of,  294 
crystalline  form  of,  382 
daily  excretion  of,  382 
elimination,  a  study  of,  574 
experiments  on,  383 
formula  for,  381 
from  creatine,  350 
JafTe's  reaction  for,  385 
quantitative  determination  of.  276,  506 
Salkowski's  test  for.  385 
separation  of.  from  urine.  383 
Weyl's  test  for.  385 
Creatinine-zinc  chloride,  formation  of,  383,  385 
Cresol,  para,  212 
tests  for,  219 
Cross  and  Sevan's  reagent,  49 
preparation  of,  49 
solubility  test,  49 
Cryoscopy,  365 
Cul-de-sac,  139 
Cupri-potassium  biuret,  formation  of.  99 

formula  for,  99 
Cyanuric  acid,  373 

formula  for,  373 
Cylindroids  in  urinary  sediments,  46s.  472 
flt-Cyprinine.  67 

Cystine,  65.  67-69.  75,  87,  458.  462 
crystalline  form  of,  76 
detection  of,  462 
formula  for,  76 


Cystine  in  hair,  87 

in  urinary  sediments,  458.  462 
preparation  of.  in  crystalline  form.  87 

Cytosine,  12s,  126,  128,  133 

Wheeler-Johnson  reaction  for,  133 

Dakin's  methods  for  quantitative  determination 

of  hippuric  acid,  520 
Dare's  hemoglobinometer,  302 
description  of.  302 

determination  of  hemoglobin  by.  302 
Deaminases.  4 

Decomposition  products  of  proteins.  63,  67,  68, 
69.  71 
crystalline  forms  of.  72-84 
experiments  on.  85-87 
isolation  of,  85-87 
Defensive  enzymes,  3 

Degradation  products  of  protein  (see  Decomposi- 
tion products).  63,  67-69,  71 
Dehn-Clark  method  for  chlorides,  558 
Delusive  feeding  experiments,  138 
Derived  proteins,  94.  114 
Detection  of  preservatives  in  milk,  323 
boric  acid  and  borates,  324 
formaldehyde,  323 
hydrogen  peroxide,  324 
salicylic  acid  and  salicvlates,  323 
Determination  of  acetoacetic  acid  in  blood,  284,  294 
in  urine.  533 
acetone  in  blood.  284,  294 

in  urine.  538 
acetone  and  diacetic  in  blood,  284,  294 

in  urine,  533 
acidity  of  urine,  by  titration.  479 

by  hydrogen  ion  concentration.  470 
albumin  in  urine.  531 
allantoin  in  urine,  518 
amino  acid  nitrogen.  88.  91.  277.  502 
in  blood.  277 

in  protein  hydrolysis.  64.  88.  91 
in  urine.  502 
of  ammonia  in  urine.  499 
amylolytic  activity.  192 
ash  of  milk,  327 
calcium  in  urine,  559 
casein  of  milk,  327-329 
catalase,  16 
chlorides  in  blood.  286 

in  urine.  556 
cholesterol  in  blood.  285 
creatine  in  blood.  276 

in  urine.  508 
creatinine  in  blood.  276 

in  urine.  506 
diacetic  acid  in  blood.  284.  294 

in  urine,  533 
fat  in  blood.  295 
in  feces,  243 
in  milk,  324 
fecal  amylase.  2i<) 
fecal  bacteria.  241 
glucose  in  urine.  522 
hippuric  acid  in  urine,  519 
hydrogen  ion  concentration  vii  i.-'..  --^a 

of  urine,  480 
^-hydroxybutyric  acid  in  blood.  284.  294 
in  urine.  SiO-  54* 


020 


INDEX 


Determination,  indican  in  urine,  542 

indole  in  feces,  242 

iron  in  urine,  591 

lactalbumin  in  milk,  329 

lactose  in  milk,  329 

magnesium  in  urine,  559 

nitrogen  in  urine,  483-491 

partition  in  urine,  484,  577 

oxalic  acid  in  urine,  545 

0-oxybutyric  acid  in  blood,  284,  294 
in  urine,  539,  541 

peptic  activity,  165 

phenols  in  urine,  543 

phosphorus  in  urine,  552 

potassium  in  urine,  561 

protein  in  milk,  327,  328 

protein  in  urine,  531 

purine  bases  in  urine,  513 

purine  nitrogen  in  urine,  516 

sodium  in  urine,  561 

sulphur  in  urine,  546 

total  solids  in  milk,  327 

total  solids  in  urine,  483 

tryptic  activity,  169 

urea  in  blood,  274 

in  urine,  491-499 

uric  acid  in  blood,  274 

in  urine,  510-513 
Deuteroprotcose,  95,  119 
Dextnn,  20,  43,  47,  56 

achroo-,  43,  56 

a-achroo-,  56 

/3-achroo-,  56 

7-achroo-,  56 

erythro-,  43.  56 

action  of  tannic  acid  on   48 

diffusibility  of,  48 

Fehling's  test  on,  48 

hydrolysis  of,  48 

iodine  test  on,  47 

solubility  of,  47 
Dextrosazone,    crystalline    form    of,     Plate    III, 

opposite  p.  22 
Dextrose  (see  Glucose) . 
Diacetic    acid    (see   Acetoacetic   acid),    270,    284. 

412,  438,  539 
Diamine  acid  nitrogen,  64 

Diaminotrihydroxydodecanoic  acid,  65,  67-69,  8s 
a-<-di-amino-caproic  acid,  68,  80 
Dialysis,  24 

Dialyzers,  preparation  of,  24 
Diastase  (see  Vegetable  amylase),  4,  10 
Diazo-benzene-sulphonic  acid,  454 

reagent,  preparation  of,  454 
Diazo  reaction  (Ehrlich's),  454 
Differentiation  between   pepsin  and   pepsinogen, 

141.  145 
Digestion,  gastric,  138 
intestinal,  195 

pancreatic,  185 
salivary,  51 
Di-iodo-hydroxypropane  (lothion),  30,  421 
Di-methyl-amino-azobenzene    (see    Topfer's    re- 
agent), 175 
2,  s-dinitrohydroquinol,  use  of,  158 
Dipeptides,  66,  95 
Disaccharides,  19,  38 
classification  of,  19 


Dissociation  products  of  protein  (see  Decom- 
position products,  63). 

Donne's  pus  test,  432 

Doremus-Hinds  ureometer,  497 

Drying  method  for  determination  of  total  solids 
in  urine,  482 

Duodenum,  epithelial  cells  of,  185 

Earthy  phosphates  in  urine,  406,  408 

quantitative  determination  of,  553 
Edestan,  94,  114 

experiments  on,  115 
Edestin,  67,  93,  109 

coagulation  of,  109 

crystalline  forms  of,  109 

decomposition  of,  67 

microscopical  examination  of,  109 

Millon's  test  on,  109 

preparation  of,  109 

solubility  of,  109 

tests  on  crystallized,  109 
filtrate  of,  no 
Ehrlich's  diazo-benzene-sulphonic     acid    reagent, 

preparation  of,  454 
Ehrlich's  diazo  reaction,  454 
Ehrlich's  mechanical  eye-piece,  use  of,  307 
Einhorn's  bead  test,  238 
Einhorn's  saccharometer,  31 
Elastin,  93,  331,  333.  334 

adsorption  of  pepsin  by,  334 

experiments  on,  334 

preparation  of,  334 

solubility  of,  334 
Electrical  conductivity  of  urine,  366 
Electrolytes,  influence  on  enzyme  activity,  7,  188 
Embryos,  glycogen  in,  341 
Emulsin,  4 

Energy  metabolism,  564,  584 
Enterokinase,  187,  196 

demonstration  of,  197 
Enzymes,  i 

activation  of,  6 

adsorption  of,  6 

classification  of,  4 

defensive,  3 

definition  of,  2 

experiments  on,  10 

influence  of  electrolytes,  7,  188 

list  of,  4 

preparation  of,  5,  11,  13,  14 

properties  of,  6 

reference  books,  10 
Epiguanine,  370,  401 
Epinepherine,  determination  of  in  adrenal  glands, 

290 
Episarkine,  370,  401 
Epithelial  cells  in  urinary  sediments,  465 

casts  in  urinary  sediments,  465,  469 
Epithelial  tissue,  330 

experiments  on,  331 
Epstein's  sugar  method,  282 

apparatus  for,  283 
Erepsin,  5,  195,  198 

experiments  on,  198 
Erythrocytes,  2^9,  261,  472 

counting  the,  304,  308 

diameter  of,  249 

form  of,  249 


INDEX 


621 


Erythrocytes,  influence  of  osmotic  pressure  on, 
261 

in  urinary  sediments,  46s,  472 

number  of,  per  cubic  millimeter,  249,  250 

of  different  species,  249 

stroma  of,  249 

variation  in  number  of,  250 
Erythro-dextrin,  43,  56 
Esbach's  albuminometer,  532 

method  for  determination  of  albumin,  532 

reagent,  preparation  of,  532 
Ester,  definition  of,  176  ^ 

hydrochloric  acid,  of  hematin,  266 

sulphuric  acid,  of  hematin,  266 
Ethereal  sulphates,  369,  385 

quantitative  determination  of,  547,  ssr 
Ethereal  sulphuric  acid,  212,  369,  385 
Ethyl  butyrate  test  for  pancreatic  lipase,  193 

sulphide,  392 
Euglobulin,  246 
Excelsin,  109 

crystalline  form  of,  no 
Extractives  of  muscular  tissue,  340 
nitrogenous,  341 
non-nitrogenous,  341 

Fasting,  feces  in,  227 

metabolism  in,  584 
Fatigue  substances  of  muscle,  34s 
Fats,  176 

absorption  of,  178,  188 

apparatus    for     determination     of     melting- 
point  of,  183 

chemical  composition  of,  176,  177 

congealing-point  of,  184 

crystallization  of,  178,  l8i 

digestion  of,  178,  188 

emulsification  of,  178,  180 

experiments  on,  180 

formation  of,  from  protein,  179-180 

formation  of  acrolein  from,  180 

hydrogenation  of,  177 

hydrolysis  of,  177 

influence  of,  on  carbohydrate  absorption,  568 

in  milk,  313,  319,  323 

in  urine,  412,  444,  465,  469 

melting-point  of,  183 

nomenclature  of,  176,  177 

occurrence  of,  176 

permanent  emulsions  of,  178,  180 

protein-sparing,  action  of,  579 

quantitative  determination  of,  in  milk,  324 

rancid.  178 

reaction  of,  178 

saponification  of,  177,  181,  182 

solubility  of,  178,  i8o- 

transitory  emulsions  of,  178,  180 
Fat-splitting   enzymes    (see   Lipases,    s,    13,    178, 

i88,  193) 
Fatty  acid,  176,  178,  182,  212,  370,  397 
Fatty  casts  in  urinary  sediments,  46s,  469 
Fatty  degeneration,  179 

Fecal  amylase,  quantitative  determination  of,  239 
Fecal  bacteria,  221,  224,  225,  241,  588 

quantitative  determination  of,  241 
Feces,  221,  587,  592 

agar-agar,  influence  of,  223-224,  589 

albumin  and  globulin  in,  237 


Feces,  bacteria  in,  221,  224,  238,  241,  588 

bacterial  nitrogen  in,  demonstration  of,  s88 

bile  acids  in,  236 

bilirubin  in,  222,  236 

blood  in,  225,  233 

carbohydrate  in,  estimation  of,  591 

casein  in,  236 

cholesterol  in,  224,  232 

collection   and   preservation   in    metabolism 
experiments,  588 

color  of,  influence  of  drugs  and  foods  upon, 
238 

daily  excretion  of,  221,  589 

enzymes  of,  226,  239 

experiments  on,  229,  587,  592 

"fasting,"  227 

fat  in,  determination  of,  243 

food  residues  in,  227,  590 

form  and  consistency  of,  223 

hydrobilirubin  in,  222,  235,  237 

hydrogen  ion  concentration  of,  223 

indole  in,  223,  237 
determination  of,  242 

influence  of  defective  mastication  on,  590 

inorganic  constituents  of,  225,  237,  591 
elements  of,  demonstration  of,  591 

macroscopic  constituents  of,  224 

metabolic  product,  nitrogen  in,  227    589 

microscopic  constituents  of,  224 

microscopical  examination  of,  229 

nitrogen  of,  227 

nucleoprotein  in,  236 

odor  of,  223 

parasites  and  ova  in,  227 

pigment  of,  222 

proteose  and  peptone  in,  237 

reaction  of,  223 

Scybala  form  of,  223 

separation  of,  importance  of,  224,  587 

separation  of,  experiment  on,  239,  587 
Fehling's  method  for  determination  of  glucose, 
in  blood,  279 
in  urine.  523 
Benedict's  modifications  of,  522 

solution,  preparation  of,  600 

test,  26,  416 

Allen's  modification  of.  419 
Benedict's  modifications  of,  27,  417 
Ferments,  classification  of,  4 
Fermentation,  lactic  acid,  170,  314 

"sugar-free,"  4,  10.  31.  421 
Fermentation  method  for  determination  of  glu- 
cose, 530 
Fermentation  test.  31,  421 
Ferric  chloride  test  for  thiocyanate  in  saliva,   59 

for  melanin  in  urine.  452 
Fibrin,  257,  268,  465,  47-4 

carmine,  preparation  of.  12 

congo-red.  preparation  of.  12 

in  urinary  sediments.  465.  474 

separation  of,  from  blood,  247,  257 

solubility  of,  268 
Fibrin  ferment,  247,  257 
Fibrin-heteroproteose,  68 
Fibrinogen,  247,  256.  257,  268 
Fibroin,  Tussah  silk,  68 
Fischer  apparatus,  75 

photograph  of,  75 


622 


INDEX 


Fleischl's  hemometer,  300 
description  of,  299 

determination  of  hemoglobin  by,  299 
Fleischl-Miescher  hemometer,  301 
Fluorides  in  urine,  370,  411 
Fly-maggots,  experiments  on,  179 
Foam  test  for  bile  acids,  208,  434 
Folin-Hart    method    for    determination    of    com- 
bined acetone  and  acetoacetic  acid,  533 
for    determination    of   acetoacetic    acid- 
539 
Folin-Messinger-Huppert  method  for  determina- 
tion of  acetoacetic  acid,  539 
Folin's    method     for    determination    of     acetone 
in  urine,  538 
acidity  of  urine,  by  titration,  479 
ammonia,  499 
creatinine,  in  blood,  276 

in  urine,  506,  508 
ethereal  sulphates,  547 
inorganic  sulphates,  547 
total  sulphates,  546 
urea,  in  urine,  495 
of  preparing  cystine,  87 
test  for  uric  acid,  381 
Folin   and    Denis'    method   for   determination   of 

uric  acid  in  blood,  274 
Folin   and    Denis'    method   for   determination   of 

uric  acid  in  urine,  modification  of,  510 
Folin  and  Denis'  method  for  phenols  in  urine,  543 
Folin  and  Denis'  method  for  urea,  49s 
Folin     and     Denis'     nephelometric     method     for 

albumin  in  urine,  533 
Folin  and  Denis'  nephelometric  method  for  ace- 
tone bodies  in  urine,  541 
Folin  and  Flanders'  method  for  hippuric  acid  in 

urine,  519 
Folin  and  Macallum's  microchemical  method  for 

ammonia,  501 
Folin  and  Pettibone's  microchemical  method  for 

urea  in  urine,  494 
Folin,    Benedict   and    Myers'    method   for   deter- 
mination of  creatine,  509 
Folin-Benedict     method     for     determination     of 

creatine,  508 
Folin-Farmer    microchemical    method    for    total 

nitrogen  in  urine,  485 
Folin-Farmer   microchemical    method.   Bock   and 

Benedict's  modification  of,  488 
Folin-Farmer    microchemical     method,     Gulick's 

modification  of,  490 
Folin-Shaffer   method   for   determination  of   uric 

acid,  SI  I 
Foods,  composition  of,  569 
purine  content  of,  572 
Foreign  substances  in  urinary  sediment,  465.  4  74 
Formaldehyde,  as  milk  preservative,  323 

reaction  (Konto),  218 
Formaldehyde-H2S04  test  (Morner),  86 
Formation  of  methyl-phenylfructosazone,  35 
Form  elements  of  blood,  245 
Formic  acid,  26,  370,  397 

Formol  titration  method  of  Benedict-Murlin,  504 
of  Malfatti,  502 
of  Sorensen,  502 
Fractional  coagulation  of  proteins,  105,  117 

method  of  gastric  analysis,  148,  159 
Free  hydrochloric  acid,  56,  140,  155 


Free  hydrochloric  acid,  tests  for,  155 
Freezing-point  of  bile,  203 
blood,  24s 
milk,  314 

pancreatic  juice,  186 
urine,  36s 
Frey-Gigon   method  for  amino-acid    nitrogen  in 

urine,  505 
Fructose,  Borchardt's  reaction  for,  35 
in  urine,  446 

methyl-phenylhydrazine  test  for,  35 
Seliwanoff's  reaction  for,  35 
Fuchsin-frog  experiment,  347 
Fuld  and  Levison's  method  for  peptic  activity, 

167 
Fundus  glands,  139 
Furfural,  formation  of,  22 

solution,  preparation  of,  600 
Fusion  mixture,  preparation  of,  129 

Galactans,  20,  so 

Galactase,  319 

Galactose,  19,  36,  314,  446 

experiments  on,  36 
Gallic  acid  test  for  formaldehyde,  323 
Ganassini's  test,  381 

Gastric  acidity  and  the  use  of  indicators,  152 
Gastric  analysis,  148,  174 

detection  of  bile  in,   171 
of  blood  in,  171 
of  food  rests  in,  173 
of  lactic  acid  in,  170 
of  mucus  in,  173 
determination  of  free  acidity  in,  164 
of  peptic  activity  in,  165 
of  total  acidity  in,  162 
of  tryptic  activity  in,  169 
examination  of  samples  in,  162 
.    fractional  method  of,  148,  159 
introduction  of  the  tube  in,  159 
Rehfuss  tube,  use  of  in,  148,  159 
retention  meal  in,  161 
test  meals  in,  161 
use  of  indicators  in,  152 
Gastric  digestion,  138 

conditions  essential  for,  14s 

general  experiments  on,  145 

influence  of  bile  on,  147 

influence   of  dififerent   temperatures   on, 

influence  of  water  on,  138 
most  favorable  acidity  for,  14s 
power  of  different  acids  in,  146 
products  of,  141,  144 

Gastric  fistula,  138 

Gastric  juice,  139 

acidity  of,  140,  151 

influence  of  water  on,  138 

of  regurgitation  on,  140,  151 
analysis  of,  148,  174 
artificial,  preparation  of,  144 
composition  of,  125,  151 
enzymes  of,  139 
lactic  acid  in,  test  for,  170 
origin  of  hydrochloric  acid  of,  140 
quantitative  analysis  of,  148,  174 
quantity  of,  138 
reaction  of,  139 


INDEX 


623 


Gastric  juice,  secretion  of,  138,  139 

influence  of  water  on,  138 
specific  gravity  of,  139 
Gastric  lipase,  139,  '43 
Gastric  protease,  12 
Gastric  rennin,  139,  143.  I47.  31S.  32i 

action  of,  upon  casein,  143,  IJ7,  315,  321 
experiments  on,  147,  32  i 
influence  of,  upon  milk,  147,  321 
in  gastric  juice,  absence  of,  143 
nature  of  action  of,  143,  315 
occurrence  of,  143 
Gastric  residuum,  160 

composition  of.  151 
Gelatin,  67.  68,  332,  33i 
coagulation  of,  333 
decomposition  of.  67,  68 
experiments  on,  333 
formation  of,  333 
Hopkins-Cole  reaction  on,  333 
Gelatin,  Millon's  reaction  on,  333 
precipitation  of,  by  alcohol,  333 
alkaloidal  reagents,  333 
metallic  salts,  333 
by  mineral  acids,  333 
preparation  of,  from  cartilage,  335 

from  collagen,  333 
salting-out  of,  333 
solubility  of,  333 
Gerhardt's  test  for  acetoacetic  acid,  439 
Gerhardt's  test  for  urobilin.  400 
Gies'  biuret  reagent,  preparation  of,  100 
Gliadin,  93,  1 11,  112 

decomposition  of,  67 
preparation  of,  112 
tests  on,  1 12 
Globin,  67,  93,  113,  250 

decomposition  of.  67 
Globulins,  93.  108 

experiments  on,  109 
preparation  of,  109 
serum,  93,  246,  412,  422 
in  urine,  412,  422 
tests  for,  423 
vegetable.  109 
Glucoproteins  (see  Glycoproteins,  pp.  94,   112) 
Glucose,  Allen's  modification  of  Fehling's  test  for, 
419 
Barfoed's  test  on,  30 
Benedict's  modification  of  Fehling's  test,  27, 

417 
Boettger's  test  on,  29,  420 
Bottu's  test  on,  23,  415 
Cipollina's  test  on,  23,  414 
Cramer's  mercuric  oxide  test  on,  28,  419 
diflFusibility  of,  24 
experiments  on,  21,  413 
Fehling's  test  on,  26,  416 
fermentation  of,  31,  421 
formula  for,  21 
glycosuria  produced  by,  568 
Haines  test  on,  419 
hyperglycemia  produced  by,  566 
iodine  test  on,  24 
Molisch's  reaction  on,  21 
Moore's  test  on,  24 
Nylander's  test  on,  29,  420 
phenylhydrazine  test  on,  22,  413 


Glucose,  quantitative  determination  of,  522 

reduction  tests  on,  25.  415 

Riegler's  reaction,  23,  414 

solubility  of,  21 

Trommer's  test  on,  25,  416 
Glucosidases,  4 
o-Glucosides,  4 
/3-Glucosides,  4 

Glucosazone,  crystalline  form  of,  Plate  III,  oppo- 
site p.  22 
Glucothionic  acid,  467 
Glutamic  acid,  69,  82,  186 

formula  for,  82 
Glutelins,  93,  no 

tests  on.  III 
Gluten,  preparation  of,  in 

tests  on.  III 
Glutenin,  93,  no,  iii 

preparation  of,  in 

tests  on,  in 
Glycerol,  176,  177,  178,  182 

borax  fusion  test  on,  183 

experiments  on,  182 

formula  for,  177 

hypochlorite-orcinol  reaction  for,  183 
Glycerol  extract  of  pig's  stomach,  preparation  of, 

144 
Glycerophosphoric  acid,  353,  354,  370,  399 
Glycocholic  acid,  204 
Glycocholic  acid  group,  204 
GlycocoU,  6s,  69,  71,  204 

crystalline  form  of,  211 

formula  for,  71,  204 

preparation  of.  211 
GlycocoU    ester    hydrochloride,    crystalline    form 

of,  72 
Glycogen,  20,  47.  341.  348 

experiments  on,  349 

hydrolysis  of,  349 

in  embryos,  341 

influence  of  saliva  on,  349 

iodine  test  on.  349 

preparation  of.  3J8 
Glycogenase,  4 
Glycolytic  enzymes.  3.  4 
Glycoproteins,  94,  112-113,  332 

experiments  on,  332 

hydrolysis  of,  332 
Glycosuria,  alimentary,  21,  568 

by  glucose  ingestion,  21,  568 
Glycosuric  acid,  395 
Glycuronates,  conjugate,  26,  417.  442 

tests  for,  442 
Glycuronic  acid,  36,  38,  442 
Glycyl-glycine.  formation  of.  70 
Glycyl-tryptophanc  test,  195,  199 
Glyoxylic  acid,  98 

formula  for,  98 
reaction  (Hopkins-Cole),  98 
Gmelin's  test  for  bile  pigments.  207,  433 

Roscnbach's  modification  of,  207,  433 
Gout,  blood  in,  248,  270,  271,  379 
Granular  casts  in  urinary  sediment,  465,  467 
Granulose,  43 

Green  stools,  cause  of,  222,  238 
Gross'  method  for  quantitative  determination  of 

tryptic  activity,  191 
Guaiac  solution,  preparation  of,  600 


624 


INDEX 


Guaiac  test  on  blood,  23s,  258,  262,  430 

on  feces,  235 

on  milk,  320 

on  pus,  432 

on  urine,  430 
Guaiac  test,  Schumm's  modification  of,  262 
Guanase,  A 

Guanidine-a-amino-valerianic  acid,  68,  79 
Guanidine-residue,  64 
Guanine,  4,  125-127,  I33.  34i 
Guanine  chloride,  crystalline  form  of,  135 
Gulick's  colorimetric  method  for  total  nitrogen  in 

urine,  490 
Gulick's  micro-oxidation  flask,  490 
Gum  arable,  20,  50,  51 

Gums   and   vegetable   mucilage   group  of   carbo- 
hydrates, 20 
Gunning's  iodoform  test  for  acetone,  436 
Gunzberg's  reagent,  as  indicator,  155 

preparation  of,  ISS 

Haine's  test  on  sugar,  419 

Hair,  human,  330 

Hammerschlag's    method    for    determination    of 

specific  gravity  of  blood,  260 
Hammarsten's  reaction,  207,  433 

reagent,  preparation  of,  207,  433 
Harding  and   MacLean's  method  for  determina- 
tion of  amino-acid  nitrogen,  91 
Hart's  casein  method,  328 
Haser's  coefficient,  364,  483 
Hayem's  solution,  601 
Hay's  test  for  bile  acids,  209,  434 
Heintz  method  for  determination  of  uric  acid,  512 
Helicoprotein,  94 

Heller's  test  for  blood  in  urine,  429 
Heller's  ring  test  for  pi-otein,  103 
Heller- Teichmann  reaction  for  blood  in  urine,  430 
Hemagglutination,  250,  262 
Hemagglutinin,  250,  262 
Hematein  test  for  blood  in  feces,  234 
Hematin,  113,  250,  254,  266 

acid-,  254,  299 

alkali-,  254,  298 

preparation  of,  266 

reduced  alkali-,  299 
Hematoidin,  205,  222,  224 

crystalline  form  of,  205,  222 

in  urinary  sediments,  458,  464 
Hematoporphyrin,  205,  254,  359,  412,  444 

in  urine,  359,  412,  444 
Hematuria,  429 
Hemicellulose,  20,  50 

experiments  on,  51 

utilization  of,  by  animals,  50 
Hemin  crystals,  form  of,  265 

tests,  264,  266,  429 
Hemiurate,  461 

Hemochromogen,  113,  250,  254,  269,  299 
Hemocyanin,  94,  113 
Hemocytometer,  Btirker's,  308 

Thoma-Zeiss,  304 
Hemoconein  (see  Blood  dust,  24s,  256) 
Hemoglobin,  94,  113,  249,  250,  254,  25S,'262,|266, 
296 

carbon  monoxide,  254,  297 

decomposition  of,  250 

derivatives  of,  relationship  of,  254 


Hemoglobin,  diffusion  of,  262 

met,  254,  298 

oxy,  250,  254,  266,  296 

quantitative  determination  of,  299-304 

reduced,  254,  296 
Hemoglobins,  94,  113 
Hemoglobinuria,  429 
Hemolysis,  245,  261 

Henderson  and  Palmer's  method  for  determina- 
tion of  hydrogen  ion  concentration,  480 
Herter's     naphthaquinone    reaction    for     indole, 

218 
Herter's   para-dimethylaminobenzaldehyde   reac- 
tion, 219 
Heterocyclic  nucleus,  65,  68,  69 
Heteroprotebse,  96  119 
Heteroxanthine,  370,  401 
Hexone  bases,  80 
Hexosans,  20 
Hexoses,  19,  20 
Hippuric  acid,  72,  369,  388,  390,  396,  585 

cystalline  form  of,  389 

Dakin's   method   for   quantitative   determi- 
nation of,  520 

experiments  on,  389,  585 

formula  for,  388 

in  urinary  sediments,  463 

Liicke's  reaction  for,  390 

melting-point  of,  389 

Roaf 's  method  for  crystallization  of,  390 

separation  of,  from  urine,  389 

solubility  of   390 

sublimation  of,  390 

synthesis  of,  390,  585 

demonstration  of,  585 
Histidine,  65,  77 

hydrochloride,  crystalline  form  of,  78 

Knoop's  color  reaction  for,  78 
Histones,  93 

Hoffmann's  reaction  for  tyrosine,  86 
Homogentisic  acid,  26,  369,  394,  417 

formula  for,  394 
Hopkin's  thiophene  reaction  for  lactic  acid,  171 
Hopkins-Cole  reaction,  98 

on  solutions,  98 
on  solids,  107 
Hopkins-Cole  reagent,  preparation  of,  98 
Hopkins-Cole    reagent    (Benedict    modification), 

preparation  of,  98 
Hordein,  93,  in 

Horismascope  (see  Albumoscope,  103,  423) 
Hormones  definition  and  discussion  of,  185,  318 

in  blood,  185 
Hiifner's  urea  apparatus,  498 
Human  fat,  composition  of,  178 

hair,  composition  of,  330 

milk,  differentiation  from  cow's,  316,  320 
Hunter  and  Givens'  modification  of  Kruger  and 

Schmidt's  method,  515 
Huppert's  reaction  for  bile  pigments,  207,  433 
Hurthle's  experiment,  349 
Hurtley's  test  for  acetoacetic  acid,  440 
Hyaline  casts  in  urinary  sediments,  465,  468 
Hydrobilirubin,  detection  of,  in  feces,  222,  23s 

extraction  of,  235 
Hydrochloric  acid  of  the  gastric  juice,  140,  ISI 
origin  of,  theories  as  to,  140 
seat  of  formation  of,  139 


INDEX 


62: 


Hydrochloric  acid  test  for  formaldehyde  (Leach), 
323 
acid-zinc  chloride  solubility  test,  49 
Hydrogen     ion     concentration     and     titratable 
acidity,  iS5,  479-480 
mode  of  expressing,  152 
of  blood,  determination  of,  288 
of  urine,  determination  of,  480 
as  influenced  by  diet,  580 
by  acids,  582,  583 
by  alkali,  582,  583 
comparison     of,      with      titratable 

acidity,  ISS.  is8 
determination     of,     by     means     of 
indicators,  158,  480 

of  McClendon's  electrode, 
149 
Hydrogen  peroxide  in  urine,  369,  411 

detection  of,  in  milk,  324 
Hydrogenated  fat,  177 
Hydrogenation,  definition  of,  177 
Hydrolysis  of  cellulose,  48,  49 
cerebrin,  357 
dextrin,  47,  48 
glycogen,  349 
inulin,  46 
proteins,  64 
starch,  43,  45 
sucrose,  41,  42 
/9-Hydroxybutyric   acid,    270,   271,  284,  295,  412, 
440,  S41 
Black's    method    for    determination    of, 

S4I 
Black's  reaction  for,  441 
formula  for,  440 
in  blood,  270,  284,  295 
origin  of,  441 

polariscopic  examination  for,  442 
quantitative  determination  of,  in  blood, 
284,  295 
in  urine,  539.  54 1 
Shaffer     and     Marriott's     method     for 
determination  of,  539 
Hyperacidity,  140,  164 

curve,  164 
Hypercholesterolemia,  247,  270,  271 
Hyperglycemia,  270,  566 

produced  by  carbohydrate  ingestion,  566 
Hypoacidity,  140 
Hypobromite  methods  for  determination  of  urea 

in  urine,  496 
Hypobromite  solution,  preparation  of,  601 
Hypochlorite-orcinol  reaction  for  glycerol,  183 
Hypoxanthine,  127,  341,  346,  350,  370,  401 
chloride,  crystalline  form  of,  136 
formula  for,  127,  346 
oxidase,  127 
Hypoxanthine  silver  nitrate,  crystalline  form  of, 
351 

Ichthulin,  94 
Ignotine,  341,  346 

formula  for,  346 
Imide  bonds,  70 
Iminazolethylamine,  214 
Iminazolpropionic  acid,  214 
Indican,  212,  386,  542 

formula  for,  212,  387 
40 


Indican,  Jaffe's  test  for,  387 

Jolles'  reaction  for,  388 

Obermayer's  test  for,  388 

origin  of,  212,  386 
Indicator  method  for  determination  of  hydrogen 
ion  concentration,  158,  480 

solutions,  preparation  of,  158 
use  of,  158 
Indicators,  experiments  on,  152 

table  of,  153,  158 

tabulation  of  results  of  tests  on,  154 

use  of,  152 

in  gastric   analysis,  152 
Indigo-blue,  213,  388 

formula  for,  213,  388 
Indigo  in  urinary  sediments,  458,  464 
Indolacetic  acid,  214,  4i2 
Indole,  212,  218,  223,  237 

formula  for,  212 

origin  of,  212 

in  feces,   quantitative  determination  of,  by 
Bergeim's  method,  242 

test  for,  218,  237 
/S-Indole-a-amino-propionic  acid,  68,  77 
Indolpropionic  acid,  214 
Indoxyl,  212 

formula  for,  212,  388 

origin  of,  212 

potassium    sulphate    (see    Indican,    p.    212, 
387.  542 
Indoxyl-sulphuric  acid,  212,  369,  385 

formula  for,  212,  386 
Influence  of  purine-free  and  high  purine  diets,  571 
Infraproteins  (see  Metaproteins,  94,  115,  116) 
Inorganic  elements  in  feces,  absorption  of,  591 

physiological  constituents  of  urine,  370,  402 
Inosinic  acid,  341,  346 

formula  for,  346 
Inositol,  19,  412,  450 

formula  for,  450 

in  urine,  412,  450 
Intestinal  digestion,  19s 

jviice,  195 

enzymes  of,  195,  196,  198-201 
preparation  of,  199-201 
Inulase,  4,  46 
Inulin,  20,  46 

action  of  amylolytic  enzymes  on,  46 

Fehling's  test  on,  46 

hydrolysis  of,  46 

iodine  test  on,  46 

reducing  power  of,  46 

solubility  of,  46 

sources  of,  46 
Inversion,  41,  43 

Invertase  (see  Sucrase,  4.  41.  ipSt  199) 
Invertases.  experiments  on,  199 
Invertin  (see  Sucrase,  4,  41,  19s,  199) 
Inverting  enzymes,  3 
Invert  sugar,  41 
Iodide  of  dextrin,  45 

of  starch,  47 
Iodine  absorption  test,  183 

test  for  starch  and  dextrin,   45.   46,   47.   49. 
SJ.  56 

for  urobilin,  400 
Iodine-sulphuric  acid  test  for  cholesterol,  a  10,  357 
lodine-zinc-chloride  reaction,  49 


626 


INDEX 


Iodoform  test  for  alcohol,  42 

for  acetone  (Lieben),  437 
lodothymol  compound,  437 
"lothion,"  30,  421 

Iron,  reduced,  influence  on  color  of  feces,  238 
in  blood,  261 

detection  of,  261 
in  bone  ash,  338 

detection  of,  338 
in  protein,  63 
in  urine,  410,  562,  591 
detection  of,  410 
determination  of,  591 
Isoleucine,  6s,  80 
Isomaltose,  19,  40 
Isopropylmetacresol  (see  Thymol) 
Isovalerianic  acid,  214 

Jacoby-Solms  method,  167 

Jaffe's  reaction  for  creatinine,  382 

Jafle's  test  for  indican,  387 

V.  Jaksch-Pollak  reaction  for  melanin,  452 

Jejunum,  epithelial  cells  of,  185 

Jolles'  reaction  for  indican,  388 

Juice,  gastric,  138,  150 

pancreatic,  185 

intestinal,  19s 

Kantor  and  Gies's  biuret  paper,  100 
Kastle's  peroxidase  reaction,  320 
Kephalin.  353,  355 
Kephyr,  40 
Keratin,  93,  112,  330 
experiments  on,  331 
solubility  of,  330 
sources  of,  330 
sulphur  content  of,  330 
Ketone,  25 
Ketose,  19 

Kidney  efficiency  test,  455 
Kjeldahl  method  for  determination  of  nitrogen, 

483 
Kjeldahl-Folin-Farmer  nitrogen  method,  485 
Knoop's  color  reaction  for  histidine,  78 
Konto's  reaction  for  indole,  218 
Koumyss,  40 

Kraut's  reagent,  preparation  of,  602 
Kreosotal,  443 

Kruger  and  Schmidt's  method  for  the  quantita- 
tive   determination    of    pu- 
rine bases,  513 
of  uric  acid,  513 
Kwilecki's  modification  of  Esbach's  method,  S32 
Kynurenic  acid,  369,  395 
formula  for,  39s 
isolation  of,  from  urine,  395 
quantitative  determination  of,  395 

Laccase,  5 

Lactalbumin,  93,  313,  318 

quantitative  determination  of,  329 
Lactase,  4,  196,  200 

experiments  on,  200 
Lactic  acid,  40,  170,  342 

ether-ferric  chloride  test  (Strauss)  for,  170 

fermentation,  40,  314 

ferric  chloride  test  (Kelling)  for,  170 

Hopkins'  thiophene  reaction  for,  171 


Lactic  acid  in  muscular  tissue,  342 
in  stomach  contents,  170 
tests  for,  170. 
Uffelmann's  test  for,  170 
Lactochrome,  319,  399 
Lacto-globulin,  313,  318 
Lactometer,  determination  of  specific  gravity  of 

milk  by,  324 
Lactosazone,  crystalline  form  of,  Plate  III,  oppo- 
site p.  22 
Lactoscope,  Feser's,  326 
Lactose,  19,  40,  318 

experiments  on,  40 
fermentation  of,  40,  318 
in  urine,  412,  444 
quantitative  determination  of,  329 
Laiose  in  urine,  451 
"Laked"  blood,  24s,  261 
Laky  blood,  261 
Lanolin,  178,  188 
Laurie  acid,  313 
Leach's  hydrochloric  acid  test  for  formaldehyde, 

323 
Lecithin,  94,  247,  353 

acrolein  test  on,  356 
decomposition  of,  354 

experiments  on,  356  ^ 

formula  for,  35/) 

microscopical  examination  of,  356 
osmic  acid  test  on,  356 
preparation  of,  356 
test  for  phosphorus  in,  356 
Lecithoproteins,  94,  114 
Legal's     reaction    for    indole,  218 

test  for  acetone,  437 
Le  Nobel  reaction  for  diacetic  acid.  439 
Lenzmann-Kober  nephelometer,  cut  of,  293 
Leucine,  67,  69,  79,  85,  87 

crystalline  form  of  impure,  463 

pure,  80 
experiments  on,  87 
formula  for,  79 
in  urinary  sediments,  463 
microscopical  examination  of,  87 
separation  of,  from  tyrosine,  86 
solubility  of,  87 
sublimation  of,  87 
Leucocytes,  245,  255 
counting  the,  306 

number  of,  per  cubic  millimeter,  255 
size  of,  25s 

variation  in  number  of,  255  * 

Leucocytosis,  255 
Leucosin,  103 

Leucyl-alanyl-glycine,  formation  of,  70 
Leucyl-glycyl-alanine,  57 
Leucyl-leucine,  formation  of,  70 
Levo-a-proline,  83 

Levulosazone,  crystalline  form  of,  Plate  III,  op- 
posite p.  22 
Levulose,  (see  Fructose),  19,  34 
Lichenin,  20,  47 
Lieben 's  test  for  acetone,  437 
Lieberkuhn's  jelly  (see  Alkali  metaprotein,  p.  116) 
Liebermann-Burchard  test  for  cholesterol,  2 10,  357 
Liebermann's  reaction,  100 
Linoleic  acid,  177 
Lipase,  gastric,  139,  143 


INDEX 


627 


Lipase,  pancreatic,  5,  13,  178,  188,  193 
experiments  on,  13,  193 
ethyl-butyrate  test  for,  193 
litmus-milk  test  for,  193 
Lipases,  s,  I3.  178,  188,  193 

autolytic,  s 

experiments  on,  13,  193 

pancreatic,  13 

vegetable,  13 
Lipemia,  blood  in,  270,  271 
Lipeses,  9 
Lipins,  353 

Lipoids  of  nervous  tissue,  353.  356 
Lipolytic  enzymes  (see  Lipases,  p.  5,  13,  178.  188, 

193) 
"Litmus-milk"  test  for  pancreatic  lipase,  193 
Long's  coefficient,  364,  483 
Lucke's  reaction  for  hippuric  acid,  390 
Lugol's  solution  preparation  of,  602 
Lymph,  245,  259 
Lysine.  68,  80,  142,  186 
Lysine  picrate,  crystalline  form  of,  81 

McClendon's  electrode,  determination  of  H  ion 

cone,  by,  149 
Magnesia  mixture,  preparation  of,  602 
Magnesium  balance,  preparation  of,592 

in  bone,  detection  of,  337,  338 
in  urine,  370,  409 

quantitative  determination  of, 
559 
phosphate  in  urinary  sediments,  458, 
464 
Malfatti's  formol  titration  method  for  ammonia  in 

urine,  502 
Maltase,  4,  57,  196,  201 
experiments  on,  201 
Maltosazone,  crystalline  form  of,  Plate  III,  op- 
posite p.  22 
Maltose,  19,  39 

experiments  on,  39 
structure  of,  39 
Marsh  apparatus,  449 
cut  of,  449 
method  for  arsenic,  448 
Marshall's  hypobromite  urea  apparatus,  498 
Marshall's  clinical  urease  method  for  estimation 

of  urea  in  urine,  493 
Mastication,    defective,    influence    of,    on    food 

residues  in  feces,  227,  590 
Mauvein,  use  of,  as  indicator,  158 
McCrudden's  method  for   determination   of   cal- 
cium, 559 
of  magnesium,  559 
McLean  and  Van  Slyke's  method  for  determina- 
tion of  chlorides  in  blood,  286 
Melanin  in  urine,  412.  451 
tests  for,  452 

urinary  sediments.  458,  465 
Melting-point  apparatus,  183 

of  fats,  determination  of,  183 
Mercuric  oxide  test  for  reducing  sugar,  28,  419 
Mercury  in  urine,  450 

detection  of,  450 
Metabolism,  564 

experiments,  565 

balance  of  income  and  outgo  in,  prepa- 
ration of,  592 


Metabolism  experiments,  collection  and  preserva- 
tion of  feces  in,  588 
urine  in,  565 
separation  of  feces  in,  587 
in  acidosis,  576 
in  fasting,  584 
in  gout,  571,  574 
influence  of  acids  on,  582 
of  alkalies  on,  582 
of  defective  mastication  on,  590 
of  digestion  on,  577 
of    fats   and   carbohydrates   as    protein 

sparers  in,  579 
of  high  calorie,  non-nitrogenous  diet  on, 

584 
indigestible,    non-nitrogenous    material 

on,  589 
of  water  on,  574 
of  acid-forming  and  base-forming  foods,  580 
of  ammonium  benzoate,  585 
of  carbohydrates,  566-568,  591,  593 
of  energy,  579.  584 
of  fat,  591 

of  inorganic  elements,  591,  593 
of   nitrogen   and   sulphur   as   influenced   by 

diet,  577 
of  proteins,  569,  590 

time  relations  of,  569 
of  purines,  571-574 
on  "salt-free"  diet,  576 
on  salt-rich  diet,  576 
relation  of  bacterial  nitrogen  of  feces  to,  588 

of  metabolic  nitrogen  of  feces  to,  589 
study  of  creatinine  elimination  in,  574 
Menstrual  blood,  257 
Messinger-Huppert  method  for  determination  of 

combined  acetone  and  diacetic  acid,  535 
Metaproteins,  94.  115  ■• 

acid,  94,  115,  116 
alkali,  94.  ii5.  116 
experiments  on,  116 
precipitation  of,  116 
sulphur  content  of,  116 
Methemoglobin,  254,  298 
Methylene  blue,  146 

reaction  (Russo),  455 
Methyl-mercaptan,  212 
Methyl  orange,  use  of,  as  indicator,  158 
red,  use  of,  as  indicator,  158 
violet,  use  of,  as  indicator,  158 
Methyl-pentose  (see  Rhamnose,  p.  19) 
Methylphenylhydrazine,  35 
Methylphenylfructosazone,  formation  of,  35 
i-methylxanthin,  370,  401 
Mett's    method    for    determination    of     peptic 

activity,  165 
Mett's  tubes,  preparation  of,  166 
Micro-organisms  in  urinary  sediments,  465,  474 
in  feces,  221,  225 
in  intestine,  212,  225 
xMilk,  313 

ash  of  human  and  cow's,  316,  317 
casein  of,  313.  3IS.  3i8 
citrates  in,  313 

composition  of  human  and  cow's,  316,  317 
detection  of  calcium  phosphate  in,  322 
lactose  in,  322 
preservatives  in,  323 


628 


INDEX 


Milk,  difference  between  human  and  cow's,   316, 
317.  320 
experiments  on,  319 
formation  of  film  on,  314,  319 
freezing-point  of,  314 
guaiac  test  on,  320 

human  and  cows,  differentiation,  316,  320 
influence  of  rennin  on,  147,  321 
isolation  of  fat  from,  323,  324 
Kastle's  peroxidase  reaction  on,  320 
lactose  in,  313,  318,  322 

crystalline  form  of,  318 
fermentation  of,  314 
microscopical  appearance  of,  315,  319 
preparation  of  casein  from,  321 
properties  of  casein  of,  318,  322 
quantitative  analysis  of,  324 
reaction  of,  314,  319 
separation  of  coagulable  proteins  of,  323 
serum,  313 

specific  gravity  of,  314,  319 
unknown  constituents  of,  313 
Millon's  reaction,  97 

reagent,  preparation  of,  97 
Mohr's  method  for  determination  of  chlorides,  558 
Molisch's  reaction,  21 
Molybdate  solution,  preparation  of,  603 
Monamino  acid  nitrogen,  64 
Monosaccharides,  19,  20 
Barfoed's  test  for,  30 
classification  of,  19 
Morner-Sjoqvist-Folin  method  for  determination 

of  urea,  495 
Morner's  reagent,  preparation  of,  86 

test  for  tyrosine,  86 
Motor  and  functional  activities  of  the  stomach, 

146  . 
Mucic  acid,  36,  40,  44s,  4J6 

test,  36,  40,  445,  446 
Mucin,  55,  s8,  94,  113 
biuret  test  on,  59 
hydrolysis  of,  59 
isolation  of,  from  saliva,  59 
Mfllon's  reaction  on,  59 
Mucins,  55,  s8,  94,  113 
Mucoid,  94,  113,  331,  332 
experiments  on,  332 
hydrolysis  of,  332 
in  urine,  397,  428 
preparation  of,  from  tendon,  332 
Mucoids,  94.  113.  331.  332 
Murexide  test,  380 
Muscle  plasma,  339,  347 

formation  of  myosin  clot  in,  339,  347 
fractional  coagulation  of,  339,  347 
preparation  of,  347 
reaction  of,  342,  347 
Muscular  tissue,  339 

ash  of,  smooth  and  striated,  345 
commercial  extracts  of,  345 
experiments  on  "dead,"  348 

"living,"  347 
extractives  of,  341,  34s 
fatigue  substances  of,  34s 
formulas  of  nitrogenous  extractives  of, 

346 
glycogen  in,  341,  348 
involuntary,  339 


Muscular  tissue,  lactic  acid  in,  340,  342,  348 
magnesium  in,  demonstration,  349 
nonstriated,  339 

phosphate  in,  demonstration  of,  349 
pigment  of,  345 

preparation  of  glycogen  from,  348 
muscle  plasma  from,  347 
Muscular  tissue,  proteins  of,  339,  348 
reaction  of  living,  342,  348 
rigor  mortis  of,  339.  340 
separation  of  extractives  from,  350 
striated,  339 
voluntary,  339 
Myohematin,  345 
Myosan,  94,  348 

formation  of,  348 
Myosin,  339,  348 

biuret  test  on,  348 
coagulation  of,  348 
preparation  of,  348 
solubility  of,  348 
Myosinogen,  339,  347 
Myristic  acid,  313 
Myristin,  177 
Myrtle  wax  (see  Bay  berry  tallow,  182) 

Nakayama's  reaction  for  bile  pigments,  207,  433 

reagent,  preparation  of,  207,  433 
a-Naphthol  reaction,  21 
Naphthoresorcinol     reaction     for     glycuronates 

(Tollens),  442 
Nencki  and  Sieber's  reaction  for  urorosein,  453 
Neosine,  341 

formula  for,  346 
Nephelometer,  Bloors,  cut  of,  291 
description  of,  290 
Lenzmann-Kober,  cut  of,  293 
Nephelometric  methods,  290,  326,  327,  533,  541 
Nephelometric,  determination  of,  acetone  bodies 
in  blood,  294 
acetone  and  diacetic  acid  in  urine,  541 
fat  in  blood,  295 
in  milk,  326 
0-hydroxybutyric  acid  in  urine,  541 
proteins  in  milk,  327 
in  urine,  533 
Nephritis,  blood  in,  248,  270,  271 
Nephrorosein  in  urine,  453 
Nervous  tissue,  353 

constituents  of,  353  , 

experiments  on  lipoids  of,  356 
lipoids  of,  353.  356 
percentage  of  water  in,  353 
phosphorized  fats  of,  353 
proteins  of,  353 
Nessler- Winkler  solution,  603 
Neurine,  212 

Neumann's  method  for  total  phosphorus,  554 
Neurokeratin,  353 
Neutral  fats,  177,  178,  180 
Neutral  olive  oil,  preparation  of,  180 
Neutral  red,  use  of,  158 
Neutral  sulphur  compounds,  369,  392 
Nippe's  hemin  test,  264 
Nitrates  in  urine,  370,  411 
Nitric  acid  test  (Heller),  103 
for  phenol,  220 
Nitric  acid-MgS04  test  (Roberts),  104 


INDEX 


629 


Nitrilase,  8 

Nitrilese,  8 

Nitrites  in  saliva,  test  for,  59 

Nitrogen,  63,  64 

forms  of  in  protein  molecule,  64 
importance  of,  in  sustaining  life,  64 
in  urine,  quantitative  determination  of,  483 
Nitrogen  distribution,  calculation  of,  484 
Nitrogen  iodide,  formation  of,  436 
Nitrogen  "lag,"  569 

"partition,"  48s.  577 
Nitrogenous  extractives  of  muscular  tissue,  341, 
3S0 
formulas  for,  346 
^-Nitrophenol,  use  of,  as  indicator,  158 
Nitroprusside  reaction  for  indole  (Legal),  218 
Nitroprusside  test  for  creatinine  (Weyl),  385 
Nitroprusside-acetic     acid     test     for     creatinine 

(Salkowski),  385 
Kitroso-indole  nitrate  test,  219 
Nitrosothymol,  formation  of  in  Heller's  test,  423 
Non-nitrogenous  extractives  of   muscular  tissue, 

341 
Non-protein  nitrogen  of  blood,  248,  270,  271 
Normal  urine,  359,  369,  37 1 

characteristics  of,  359 
constituents  of,  369,  371 
experiments  on,  373-410 
Novaine,  341 

formula  for,  346 
Nubecula,  396,  428 
Nucleases,  5 

experiments  on,  134 
Nucleic  acid,  124-128,  131-137 
decomposition  of,  124-126 
experiments  on,  131— 137 
from  yeast,  formula  for,  125 
Nucleicacidase,  s»  126 
Nucleins,  123,  124,  144,  571 
Nucleoproteins,  94,  112,  123,  128-131,  396,  428 
decomposition  of,  123-124 
experiments  on,  128-131 
from  yeast,  128 

preparation  of,  128 

protein,    carbohydrate    and    phosphoric 

radicals  in,  130 
tests  on,  129 

thymus,  preparation  of,  130 
experiments  on,  130 
in  bile,  203,  207 
in  feces,  236 
in  nervous  tissue,  353 
in  urine.  369,  396,  412,  428 

test  for,  428 
occurrence  of,  123 
Ott's  precipitation  test  for,  428 
Nucleosidase,  s,  126  ' 
Nucleoside,  125 
Nucleotidase,  s,  126 
Nucleotide,  125 
Nylander  reaction,  29.  420 
Nylander  reagent,  preparation  of,  29,  420 

Obermayer's  test  for  indican,  388 
reagent,  preparation  of,  388 
Oblitine,  341 

"Occult"  blood  in  feces,  225,  233 
tests  for,  233 


Olein,  177.  313 
Olive  oil,  180 

emulsification  of,  180 
neutral,  preparation  of.  180 
Oliver's  peptone  test  for  bile  acids,  209,  433 
Optical  methods,  31-34 
Orcinol-HCl  reaction  (Bial).  37,  443 
Orcinol  test,  38,  444 

Organic  physiological  constituents  of  urine,  369 
Organized  ferments,  i 
Organized  urinary  sediments,  457,  465 
Ornithine,  63,  214 

Ortho-tolidin  test  for  blood,  171,  233,  263,  430 
Osbome-Folin  method  for  determination  of  total 

sulphur  in  urine,  349 
Ossein,  336 

preparation  for,  336 
Osseoalbumoid,  336 
Osseomucoid,  94,  113,  336 

chemical  composition  of.  113 
Osseous  tissue.  336 

experiment  on,  337 
Ott's  precipitation  test  for  detection  of  nucleo- 

protein  in  urine.  428 
Ovalbumin,  93 
Ovoglobulin,  93 

Oxalated  plasma,  preparation  of,  268 
Oxalic  acid,  369.  39i.  343 

formula  for,  391 

in  urine,  369.  391 

quantitative  determination  of,  543 
Oxaluria,  391 
Oxaluric  acid.  369,  397 
Oxamide.  99 
Oxidases,  3,  14,  319 

experiments  on,  14 
Oxy acids.  212.  217.  220.  369,  394 

tests  for.  220 
^-Oxybutyric    acid    (see   /3-hydroxybutyric    acid, 

270.  271.  284,  293,  412,  440,  341) 
Oxyhemoglobin,  64,  250,  234.  233 

Reichert's  method  for  crystallization  of,  266 

crystalline  forms  of,  231-234 
Oxymandelic  acid.  369.  395 
Oxyproline,  63,  67.  83 
Oxyproteic  acid,  369.  392.  434 

Palmitic  acid,  176,  177.  181.  182 
crystalline  form  of.  182 
experiments  on.  182 
formula  for.  177 
preparation  of.  182 
Palmitin.  176,  319 
Pancreatic  amylase,  4.  11,  187,  192 

digestion  of  dry  starch  by.  18S.  192 

inulin  by.  192 
experiments  on.  ii.  191 
influence  of  bile  upon  action  of,  192 
most  favorable  temperature  for  action 
of.  193 
Pancreatic  digestion,  183 

general  experiments  on.  190 
products  of.  186.  189 
Pancreatic  insufficiency,  Schmidt's  nuclei  test  for. 

237 
Pancreatic  juice.  123,  185,  186,  187 

artificial,  preparation  of.  189 
daily  excretion  of,  186 


630 


INDEX , 


Pancreatic  juice,  enzymes  of,  186 
freezing-point  of,  186 
mechanism  of,  secretion  of,  185 
reaction  of,  186 
solid  content  of,  186 
specific  gravity  of,  186 
Pancreatic  lipase,  5,  13,  178,  188 
experiments  on,  13,  193 
ethyl-butyrate  test  for,  193 
litmus-milk  test  for,  193 
Pancreatic  protease  (see  Trypsin,  pp.  5,  12,  186) 
Pancreatic  rennin,  5,  189,  194 
experiments  on,  194 
Papaih,  5,  13 
Papayotin  (see  Papain) 
Paracasein,  315 

Para-cresol-sulphuric  acid,  369,  385 
Paradimethylamino  benzaldehyde  solution,  prep- 
aration of,  604 
Paralactic  acid,  247,  314,  342,  370,  398 
Paramyosinogen,  339 
Paranucleoprotagon,  353,  356 
Paraoxyphenylacetic  acid,  212,  214,  369,  394 
Paraoxy-/3-phenyl-a-amino-propionic  acid,  69,  74, 

86 
Paraoxyphenylpropionic  acid,  212,  214,  369,    394 
Paraphenylenediamine  hydrochloride,  324 
Parasites,  227,  465,  474 
Paraxanthine,  370,  401 
Parietal  cells,  139 
Parotid  glands,  characteristics  of  saliva  secreted 

by,  54 
Pathological  constituents  of  urine,  412 
Pathological  urine,  359,  412 
constituents  of,  412 
experiments  on,  413 
"Partition"  of  nitrogen  and  sulphur  in  urine,  577 
Pektoscope,  365 
Pentamethylenediamine,  214 
Pentapeptides,  66,  95 
Pentoses,  19,  37 

experiments  on,  37 
in  urine,  412,  443 
tests  for,  443 
Pentosans,  20,  36,  50 

Pepsin  (see  Gastric  Protease),  S,  12,  141,  165 
action  of,  influence  of  bile  upon,  147 

influence  of  diflferent  acids  upon,  146 
influence  of  metallic  salts  upon,  146 
temperature  upon,  145 
conditions  essential  for  action  of,  14s 
differentiation  of,  from  pepsinogen,  141,  145 
digestive  properties  of,  141 
formation  of,  141 

most  favorable  acidity  for  action  of,  i43 
presence  of,  in  intestine,  142 
proteolytic  action  of,  141 
Pepsin-hydrochloric  acid,  145 
Pepsin-rennin  controversy,  143 
Pepsinogen,  6,  141,  144 

differentiation  of,  from  pepsin,  141,  145 
formation  of,  141 
extract  of,  preparation  of,  144 
Peptases,  5 

Peptic  activity,  Fuld  and  Levison's  method  for, 
determination  of,  167 
Mett's  method  for  the  determination  of, 
165 


Peptic  activity,  Rose's  method  for  determination 

of,  167 
Peptic  proteolysis,  141 

products  of,  141 

relation  of,  to  tryptic  proteolysis,  142 
Peptides,  66,  71,  95,  120 
Peptone,  6s,  7i.  95.  "8 
ampho,  95,  119 
anti,  119 

differentiation  of,  from  proteoses,  119 
experiments  on,  119,  120 
in  urine,  412,  426 
tests  for,  427 
separation  of,  from  proteoses,  120 
Peptone  test  for  bile  acids  (Oliver),  209,  435 
Periodide  test  for  choline,  357 
Peroxidases,  5,  14,  is,  321 
Peters'  method  for  sugar  determination,  525 
Pettenkofer's  test  for  bile  acids,   208,  434 

Mylius's  modification  of,   208, 

434 
Neukomm's     modification     of, 
209,  435 
Phenaceturic  acid,  370,  398 
Phenol,  213,  387 

excretion  of,  387,  545 

quantitative  determination  of,  in  urine,  543 
tests  for,  219 
Phenolphthalein  as  indicator,  153,  157,  158,  162, 
479 
preparation  of,  158,.  605 
test  for  blood  in  feces,  234 
Phenolsulphonephthalein    test    for    kidney    effi- 
ciency, 455 
Phenol-sulphuric  acid,  369,  385 
Phenylacetic  acid,  214 
Phenyl-a-amino  propionic  acid,  69,  73 
Phenylalanine,  65,  67,  69,  73 
Phenylethylamine,  214 
Phenylglucosazone,  22  and  plate  opposite 
Phenylhydrazine,  22 

acetate  solution,  preparation  of,  23 
mixture,  preparation  of,  22 
reaction,  22,  23,  413 

Cipollina's  modification  of,  23,  414 
Phenyllactosazone,  crystalline  form  of,  Plate  III, 

opposite  p.  22 
Phenylmaltosazone,  crystalline  form  of  Plate  III, 

opposite  p.  22 
Phenylpotassium  sulphate,  386 
Phenylpropionic  acid,  214 
Phloroglucinol-HCl  reaction,  36.  38,  443.  446 
Phosphate  solutions  and  hydrogen  ion  concen- 
tration, 154,  156,  157.  159.  361,  481 
Phosphates  in  urine,  361,  406,  553 
detection  of,  408 
experiments  on,  408 
quantitative  determination  of,  552 
Phosphatase,  9 
Phosphatese,  9 
Phosphatides,  203,  296,  353 
Phosphocarnic  acid,  341,  346,  370,  399 
Phosphonuclease,  136 
Phosphoproteins,  94,  95,  113,  318 
Phosphorized  compounds  in  urine,  370,  399 
Phosphorus  in  urine,  determination  of,  552 

organic,  test  for,  129 
Phosphotungstic  acid  reaction  (Folin),  381 


INDEX 


631 


Phosphotungstic    precipitation  test  for   proteose 

(v.  Aldor),  427 
Physiological  constituents  of  urine.  369 
Phytase,  s 
Phytin,  s 

Picric  acid  reaction  for  creatinine  Qaffe),  385 
Pigments  of  urine,  359.  370,  399i  4Si 
Pine  wood  test  for  indole,  219 
Piria's  test  for  tyrosine,  86 
Plasma  of  blood,  245,  268 

of  muscle,  339,  347 
Plasmaphaeresis,  249 
Polariscope,  use  of,  31 

in  detection  of  conjugate  glycuronates, 

443 
in  determination  of  dextrose,  31,  531 
0-hydroxybutyric  acid,  442,  S4i 
Polypeptides,  66,  71,  95 
Polysaccharides,  20,  43 
classification  of,  20 
properties  of,  43 
Posner's  modification  of  biuret  test,  100 
Potassium  in  urine,  370,  409 

quantitative  determination  of,  562 
Potassium   hydroxide    test    for     blood   in   urine 
(Heller),  429 
indoxyl-sulphate  (see  Indican,  pp.   212,  387, 
S42) 
formula  for,  212,  387 
origin  of,  212,  386 
tests  for,  387 
Primary  protein  derivatives,  94,  114 
Primary  proteoses,  119 
Products  of  protein  hydrolysis,  66,  67,  71 
Prolamins,  93,  ill 

classification  of,  93 
preparation  of,  112 
tests  on,  112 
Proline,  65,  67,  68,  83,  ill,  142,  186 

crystalline  form  of  copper  salt  of,  84 
crystalline     form     of    laevo-a-,  84 
Prosecretin,  185 
Protagon,  353,  354 

preparation  of,  356 
structure  of,  355 
Protamines,  classification  of,  93,  93 
Proteans,  94,  114 
Protease,  gastric,  5,  12,  141 

experiments  on,  12,  144 
pancreatic,  5,  12,  186 

experiments  on,  12,  189 
vegetable,  5,  13 
Proteases,  5,  12 

experiments  on,  12 
Protective  enzymes  (see  defensive  enzymes),  3 
Protein  content  of  foods,  569 

derivatives,  primary,  65,  94,  114 

secondary,  65,  94,  118 
metabolism,  time  relations  of,  569 

influence  of  water  on,  575 
utilization,  determination  of,  590 
Protein-coagulating  enzymes,  s.  i43.  189,  256.  315 
Protein-cystine,  77 
Protein-sparing  action  of  fat  and  carbohydrate, 

579 
Proteins,  63,  93,  412,  422 

acetic  acid  and  potassium  ferro-cyanide  test 
for,  104 


Proteins.  Acree-Rosenheim  test  on,  100 
action  of  alkaloidal  reagents  on,  103 

of  metallic  salts  on,  103 

mineral  acids,  alkalies  and  organic  acids 
on,  103 
Bardach's  reaction  on,  loi 
biuret  test  on,  98 
chart  for  use  in  review  of,  121 
chemical  composition  of,  63 
classification  of,  93,  95 
coagulated,  94,  116 

biuret  test  on,  118 

formation  of,  116 

Hopkins-Cole  reaction  on,  118 

Millon's  reaction  on,  118 

solubility  of,  118 

xanthoproteic  reaction  on,  118 

quantitative  determination  of,  327 
coagulation,  influence  of  salts  upon,  117 
coagulation  or  boiling  test  for,  105 
color  reactions  of,  97 
conjugated,  94,  93,  112,  123 

classes  of,  94,  112 

experiments  on,  59,  113,  128,  296,  321 

nomenclature  of,  94,  112 

occurrence  of,  94,  112 
decomposition  of,  64,  67,  68 

by  hydrolysis,  63 

by  oxidation,  64 

products  of,  64,  67,  68,  71 
experiments  on,  85 
separation  of,  85 
study  of,  64,  67,  85 
derived,  94,  114 
formation  of  fat  from,  179 
formulas  of,  64 
Heller's  ring  test  on,  103 
Hopkins-Cole  reaction  on,  98 
importance  of,  to  life,  63 
in  urine,  412,  422 

determination  of,  331 

test  for,  423 
Liebermann's  reaction  on,  100 
Millon's  reaction  on,  97 
of  milk,  315,  318 
molecular  weights  of,  64 
Posner's  reaction  on,  100 
precipitation  of,  by  alcohol,  106 

alkaloidal  reagents,  103 

metallic  salts,  103 

mineral  acids,  103 
precipitation  reactions  of,  102 
quantitative  determination  of,  in  milk,  328 
review  of,  121 
Robert's  ring  test  on,  104 
salting-out  experiments  on,  103 
salts  of,  103 

scheme  for  separation  of,  122 
simple,  93,  95 
synthesis  of.  66,  70 
xanthoproteic  reaction  on,  98 
Proteolysis,  peptic,  142 

tryptic.  142,  186 
Proteolj-tic  enzymes  (see  Proteases,  p.  13) 
Proteose,  64,  94,  93.  118 

V.  Aider's  method  for  detection  of,  437 
biuret  test  on,  i30 
coagulation  test  on,  120 


632 


INDEX 


Proteose,  deutero,  94,  95,  120 

differentiation  of,  from  peptone,  120 
experiments  on,  119,  120 
hetero,  95,  120 
in  urine,  412,  426 
test  for,  427 
potassium  ferrocyanide   and   acetic  test   on, 

120 
powder,  preparation  of,  120 
precipitation  of,  by  nitric  acid,  120 
by  picric  acid,  120 
by  potassio-mercuric  iodide,  120 
by  trichloracetic  acid,  120 
primary,  120 
proto,  95,  96,  120 

Schulte's  method  for  detection  of,  427 
secondary,  120 

separation  of,  from  peptones,  120 
Protoproteose,  95,  96,  120 
Proteoses  and  peptones,  95,  96,  119,  120 
separation  of,  120 
tests  on,  120 
Proteose-peptone,  119 

Proteose-peptone,  coagulation  test  on,  120 
experiments  on,  119 
Millon's  reaction  on,  119 
precipitation  of,  by  nitric  acid,  120 
by  picric  acid,  120 
Prothrombin,  256,  257 
Pseudo-globulin,  246 

Ptomaines  and  leucomaines  in  urine,  370,  401 
Ptyalin  (see  Salivary  amylase,  i,  4,  10,  55,  187) 
Purinases,  127,  134 

experiments  on,  134 
Purine  bases,  125,  126,  132,  346,  370,  401 
formulas  for,  127 
in  urine,  quantitative  determination  of,  513 

tests  on,  132,  133 
content  of  foods,  572 
excretion,  rate  of,  574 
oxidases,  s,  127,  134 
Purines,  amino,  127,  132 

oxy,  127,  132 
Purine-free  and  high  purine  diets,  influence  of,  571 
Pus  casts  in  urinary  sediments,  465,  472 
cells  in  urinary  sediments,  472 
in  urine,  431 

tests  for,  431,  432 
Putrefaction,  control  of,  by  carbohydrate,  213 

indican  as  an  index  of,  212,  386 
Putrefaction  mixture,  preparation  of  a,  214 
products,  212 

experiments  on,  214 
most  important,  212 
tests  for,  218 
Putrescine,  212 
Pyloric  glands,  138 
Pyrimidine  bases,  125,  126,  128 
experiments  on,  133 
formulas  for,  128 
Pyrocatechol-sulphuric  acid,  369,  386 
a-pyrrolidine-carboxylic  acid  (see  Proline,  pp.  65, 

67,  68,  83,  III,  142,  186) 
Pyuria,  431 

Quadriurate,  461 

Qualitative  analysis  of  the  products  of  salivary 
digestion,  60 


Quantitative  analysis  of  blood,  270 

of  gastric  juice,  148 

of  milk,  324 

of  urine,  479 
Quevenne   lactometer,    determination   of   specific 
gravity  of  milk  by,  324 

RafEnose,  19,  42 

Raiziss  and  Dubin's  volumetric  methods  for  total 

sulphur,  551 
Rancid  fat,  178 

Raw  and  heated  milk  tests,  320 
Reaction  of  the  urine,  361,  479,  580 
Reagents  and  solutions,  594-610, 
Reduced  alkali-hematin,  299 
Reduced  hemoglobin,  296 
Reductases,  319 
Rehfuss  stomach  tube,  cut  of  IJ9 

use  of,  149,  159 
Reichert's    method    for    crystallization    of    oxy- 
hemoglobin, 266 
Reinsch  test  for  arsenic,  449 

for  mercury,  450 
Remont's  method  for  detection  of  salicylic  acid 

and  salicylates,  323 
Rennin,  gastric,  139,  143,  147,  315,  321 

action  of,  upon  casein,  143,  147,  315,  321 

experiments  on,  147,  321 

influence  of,  upon  milk,  147,  321 

in  gastric  juice,  absence  of,  143 

nature  of  action  of,  143,  315 

occurrence  of,  143 
Rennin,  pancreatic,  s,  189,  194 

experiments  on,  194 
Rennin-pepsin  controversy,  143 
Resorcinol-HCl  reaction,  35,  447 
Respiration,  chemistry  of,  255 
Retention  meal  in  gastric  analysis,  161 
Reticulin,  112 

Reversibility  of  enzyme  action,  8,  57 
Reynolds-Gunning  test  for  acetone,  438 
Rhamnose,  19,  38 

Rhubarb,  influence  of,  on  color  of  feces,  238 
Ricin,  13,  262 
Riegler's  reaction,  23,  414 
Rigor  mortis,  339,  340 
Ring  test  for  urobilin,  400 
Roaf's  method  for  crystallizing  hippuric  acid,  390, 

587 
Robert's  ring  test  for  protein,  104.  424 

reagent,  preparation  of,  104,  424 
Robin's  reaction  for  urorosein,  453 
Rosolic  acid,  use  of,  as  indicator,  158,  513 
Rosenheim's  bismuth  test  for  choline,  358 
Rosenheim's  periodide  test  for  choline,  357 
Rosenheim  and  Drummond's  volumetric  methods 

for  sulphates  and  total  sulphur,  550-551 
Rose's  method  for  determination  of  pepsin,  167 
Rothera's  reaction  for  acetone,  438 
Rubner's  test  for  lactose  in  urine,  445 
Ruhemann's  uricometer,  cut  of,  513 

use  of,  5 13 
Russo's  reaction,  455 

Ruttan  and  Hardisty's  orthotolidin  test  for  blood, 
171.  233,  263.  430 

Saccharide  group,  20 
Saccharose  (see  Sucrose,  19,  41) 


INDEX 


633 


Sahli's  desmoid  reaction,  146 

reagent,  164 
Salicyl aldehyde  reaction  for  acetone  (Frommer), 

438 
Saliva,  S4 

alkalinity  of,  55 
amount  of,  55 
bacteria  in,  58 
biuret  test  on,  58 
calcium  in,  sp 
chlorides  in,  59 
constituents  of,  ss 
digestion  of  dry  starch  by,  60 
digestion  of  inulin  by,  60 
digestion  of  starch  paste  by,  56,  59 
dilution  of,  influence  on  digestion,  60 
enzymes  contained  in,  55 
excretion  of  potassium  iodide  in,  61 
inorganic  matter  in,  tests  for,  59 
Millon's  reaction  on,  58 
mucin  from,  preparation  of,  59 
nitrites  in,  test  for,  59 
phosphates  in,  test  for,  59 
potassium  thiocyanate  in,  59 
reaction  of,  ss,  58 
secretion  of,  S4 
specific  gravity  of,  55,  s8 
sulphates  in,  test  for,  S9 
tests  on,  58 
thiocyanates  in,  ss,  S9 
tripeptide-splitting  enzymes  in,  57 
Salivary  amylase,  i,  4,  10,  55.  187 

activity  of,  in  stomach,  57,  188 
inhibition  of  actixaty  of,  57 
nature  of  action  of,  s6,  57 
products  of  action  of,  56 
scheme  showing,  56 
Salivary  digestion,  54 

graphic  representation  of,  56 
influence  of  acids  and  alkalis  on,  56,  61 
dilution  on,  57,  60 
metallic  salts  on,  6x 
temperature  on,  60 
nature  of  action  of  acids  and  alkalis  on,  6i 
qualitative  analysis  of  products  of,  60 
Salivary  digestion  in  stomach,  57,  188 
glands.  54 
stimuli,  54 
Salkowski-Autenrieth-Barth    method    for    deter- 
mination of  oxalic  acid  in  urine,  54s 
Salkowski's  method  for  determination  of  purine 

bases,  516 
Salkowski-Schippers  reaction  for  bile  pigments, 

208,  433 
Salkowski's  test  for  cholesterol,  210,  357 

for  creatinine,  385 
Salmine,  bi,  68,  69,  94,  95 
"Salt-free"  diet,  metabolism  on  a,  576 
Salted  plasma,  preparation  of,  268 
Salting-out  experiments  on  proteins,  105,  122 
Santonin,  influence  of  on  color  of  feces,  238 
Saponification,  177,  181,  182 
of  bayberry  tallow,  181 
of  lard,  182 
Sarcolactic  acid,  342 

Scallops,  preparation  of  glycogen  from,  348 
Schalfijew's  method   for  preparation  of    hemin, 
366 


Schema  for  "blood  counting,"  311 
Scheme  for  analysis  of  biliary  calculi,  209 
bone  ash,  338 
stomach  contents,  159 
urinary  calculi,  477 
separation  of  carbohydrates,  53 
of  proteins,  122 
Scherer's  coagulation  method  for  determination 

of  albumin  in  urine,  531 
Schifl's  reaction  for  cholesterol,  210,  357 

for  uric  acid,  381 
Schmidt  diet,  composition  of,  228 
■  Schmidt's  nuclei  test  for  pancreatic  insufficiency, 
237 
Schmidt's  test  for  hydrobilirubin,  23s 
Schulte's   method   for   detection   of   proteose   in 

urine,  427 
Schumm's  modification  of  the  guaiac  test,  262 
Schutz's  law,  statement  of,  9,  166 
Schweitzer's  reagent,  action  of,  on  cellulose,  49 

preparation  of.  49 
Scleroproteins,  (see  Albuminoids),  93.  112 
Scombrine,  67.  94 
Scombrone,  93.  95 

Scott-Wilson  apparatus,  cut  of,  536 
Scott- Wilson  method  for  acetone  and  diacetic  acid 
in  blood.  284 
in  urine,  536 
reagent  537 
Scybala,  50,  223 

Secondary  protein  derivatives,  65,  94.  "8 
Secondary  proteoses,  120 
Secretin,  185 
SeliwanoS's  reaction,  35,  447 

reagent,  preparation  of,  35,  447 
Senna,  influence  of,  on  color  of  feces,  238 
Separation  of  feces,  importance  of,  in  nutrition 

and  metabolism  experiments,  224,  587 
Serine,  65.  67.  69,  73 

crystalline  form  of,  "3 
formula  for,  73 
Serum  albumin,  93,  245,  268,  412.  422 
in  urine,  412,  422 
test  for,  423 
Serum,  blood,  24s.  267 

milk.  313 
Serum  globulin,  93,  246,  412,  422 
in  urine,  412,  422 
test  for,  423 
Shackell's  method  for  vacuum  desiccation,  482 
Shaffer  and  Marriott's  method  for  acetone  and 

diacetic  acid,  539 
Shaffer  and  Marriott's  method  for  determination 

of  ^-hydroxybutj-ric  acid,  539 
Sherrington's  solution,  preparation  of,  30S 
Silicates  in  urine.  370.  411 
Silver  reduction  test  for  uric  acid  (Schiff),  381 
Skatole,  212,  213,  214,  219 

tests  for,  219 
Skatole-carbonic  acid.  217.  220 

test  for.  220 
Smith's  test  for  bile  pigments,  208.  433 
Soap,  salting-out  of,  181 

insoluble,  preparation  of,  182 
Sodium  and  potassium  in  urine,  370,  409.  S6i 

quantitative  determination  of,  561 
Sodium  alizarin  sulphonate  as  indicator,  153.  IS4. 
157.  158,  174,  481 


634 


INDEX 


Sodium  alizarin  sulphonate,  preparation  of,  154, 

158 
Sodium  chloride,  crystalline  form,  267 
Sodium  chloride  in  urine,  409,  556,  561,  576 
Sodium  hypobromite  solution,  preparation  of,  601 
Sodium  nitrite-ferrous  sulphate  reaction  for  di- 

acetic  acid  (Hurtley),  440 
Sodium  nitroprusside  test  for  acetone,  437 
Sodium  sulphide  solution,  preparation  of,  607 
Solera's  reaction  for  detection  of  thiocyanate  in 
saliva,  59 
test  paper,  preparation  of,  59 
Soluble  starch,  11,  43,  56 

as  indicator,  165,  280,  286,  524,  533 
Sorensen's   formol   titration   method   for   amino 
nitrogen,  502 
indicator  method  for  hydrogen  ion  concen- 
tration, 158,  480 
Soxhlet  apparatus  for  extraction  of  fat,  326 
Soxhlet    lactometer,    determination    of    specific 

gravity  of  milk  by,  324 
Specificity  of  enzyme  action,  7 
Spectroscope,  use  of  in  detection  of  blood,  297 
Spermatozoa  in  urinary  sediments,  473 

microscopical  appearance  of  human,  474 
Spiegler's  ring  test  for  protein,  104,  424 

reagent,  preparation  of,  104,  424 
Spiro's  reaction  for  hippuric  acid,  390 
Spongin,  69 

Standard  ammonium  thiocyanate  solution,  prepa- 
ration of,  594 
creatinine  solution,  599 
iodine  solution,  602 
potassium  permanganate,  605 
picramic  acid,  60s 

silver  nitrate  solution,  preparation  of,  606 
sodium  alcoholate,  607 
sodium  thiosulphate,  604,  607 
uranium  acetate  solution,  preparation  of,  609 
uric  acid  solution,  610 
Starch,  20,  43 

action  of  alcohol  on  iodide  of,  45 
action  of  alkali  on  iodide  of,  45 

heat  on  iodide  of,  46 
dry,    digestion   of,    by   pancreatic    amylase, 

188,  192 
dry,  digestion  of,  by  salivary  amylase,  60 
experiments  on,  43 
hyperglycemia  produced  by,  567 
iodine  test  for,  45 

ftiicroscopical  characteristics  of,  43,  44 
microscopical  examination  of,  45 
potato,  preparation  of,  43 
soluble,  ir,  43,  56 
soluble  starch   as  indicator,    165,    280,    286, 

524,  533 
solubility  of,  43 
various  forms  of,  44 
Starch  group,  20 

Starch  paste,  action  of  tannic  acid  on,  45 
diffusibility  of,  45 

digestion  of,  by  pancreatic  amylase,  188 
191 
by  salivary  amylase,  10,  56,  59 
Fehling's  test  on,  45 
hydrolysis  of,  45 
iodic  acid  paper,  59 
preparation  of,  45 


Steapsin  (see  Pancreatic  lipase,   5,   13,   178,   188, 

193 
Stearic  acid,  177,  354 
Stearin,  177,  178,  313 
Stellar  phosphate,  322,  j6o 
Stercobilin,  222 
Stokes'  reagent,  action  of,  296 

preparation  of,  296 
Stomach  contents,  lactic  acid  in  tests  for,  170 
examination  of,  159 
pepti de-splitting  enzyme  in,  142,  199 
removal  of,  161 
tube,  Rehfuss,  149 
Stomach,  motor  and  functional  activities  of,  159 
Stone-cystine,  77 
Sturine,  67,  68,  93 
Sublingual     glands,     characteristics     of     saliva 

secreted  by,  54 
Submaxillary    glands,    characteristics    of    saliva 

secreted  by,  54 
Substrate,  3 
Succinic  acid,  214 
Sucrase,  4,  14,  ips.  200 

experiments  on,  14,  200 
vegetable,  14 
Sucrose,  19,  41 

experiments  on,  42 
inversion  of,  42 
production  of  alcohol  from,  42 
structure  of,  41 
Sucrose-H2S04  test  (Pettenkofer),  208,  434 
Sulphanilic  acid,  455 
Sulphates  in  saliva,  test  for,  59 
Sulphates  in  urine,  370,  403,  546 
ethereal,  404,  551 

quantitative  determination  of,  547 
experiments  on,  404 
inorganic,  404,  550 

quantitative  determination  of,  547 
total,  quantitative  determination  of,  546, 
551 
Sulphocyanides   (see  Thiocyanates,   55,   59.   369, 

392) 
Sulphur  in  protein,  63,  108 
acid,  io8 
in  urine,  gravimetric  determination  of,  546 

volumetric  determination  of,  550 
lead  blackening,  108 
loosely  combined,  108 
mercaptan,  108 
neutral,  108,  404 
oxidized,  108 
"partition,"  in  urine,  577 
tests  for,  108 
unoxidized,  108 
Sulphuric  acid  test  (Piria),  86 
Surface  tension  test  for  bile  acids  (Hay),  209,  434 
Suspension  of  manganese  dioxide,  513 
Synthesis  of  hippuric  acid,  585 

demonstration  of,  585 

Tallow  bayberry,  saponification  of,  181 
Tallquist's   hemoglobin   scale,    determination   of 

hemoglobin  by,  304 
Tannic  acid,  influence  of,  on  dextrin,  48 
on  starch,  45 

precipitation  test  for  proteose  (Ott), 
429 


INDEX 


635 


Tannin  test  for  carbon    monoxide  hemoglobin, 

298 
Tanret's  reagent,  preparation  of,  104,  425 
Tanret's  test,  104,  425 
Tartar,  formation  of,  55 
Taurine,  204,  210,  341,  346,  369,  392 
derivatives.  369,  392 
formula  for,  204 
microscopical  appearance,  211 
preparation  of,  210 
Taurocholic  acid,  204 

group,  204 
Teichmann's     crystals,     form     of     (see     Hemm 
crystals,  p.  265) 
test.  264,  266,  429 
Tendomucoid,  94.  113.  332 
biuret  test  on,  332 
chemical  composition  of,  113 
hydrolysis  of,  332 

loosely  combined  sulphur  in.  test  for,  332 
preparation  of.  332 
solubility  of.  332 
Test  meals.  i6i 

Ewald,  161 
water.  i6r 
retention.  i6r 
Tetrapeptides,  66.  95 
Tetramethylene-diamine,  214 
Tetranucleotide,  123 

Thiocyanates  in  saliva,  significance  of.  53 
ferric  chloride  test  for.  39 
Solera's  reaction  for.  59 
Thiocyanates  in  urine,  369.  392 
Thiophene  reaction.  171 
Thoma-Zeiss  hemocj'tometer,  304 
Thrombin,  247.  257 
Thromboplastine,  257 
Thymol,  formula  for,  367 

interference  in  Heller's  ring  test,  423 

determination  of  sugar,  acetone  bodies, 
phosphates  and  magnesium  in  urine, 
367 
interference  of,  in  Lieben's  acetone  test,  437 
vise  of,  as  preservative,  367 
Thymolphthalein,  use  of,  as  indicator,  137,  138 
Thymus  histone.  93 
Thymus  nucleic  acid.  124.  131 

preparation  of.  131 
tests  on,  132 
Time  relations  of  protein  metabolism,  369 
Tincture  of  iodine,  preparation  of.  608 
Tissue,  adipose,  experiments  on,  176.  338 
connective,  330 

white  fibrous.  331 

composition  of,  331 
experiments  on,  332 
yellow  elastic,  333 

composition  of,  334 
experiments  on.  334 
epithelial,  330 

experiments  on  331 
muscular,  339 

experiments  on.  347 
nervous.  353 

experiments  on.  336 
osseous.  336 

experiments  on,  337 
Tissue  debris  in  urinar>'  sediments,  463,  473 


Titanium  tetrachloride  as  cellulose  solvent,  49 
Toison's  solution,  preparation  of,  608 
o-Tolidin  test  for  blood,  233.  263,  430 
ToUen's  reaction  for  conjugate  glycuronates,  442 
arabinose.  37 
galactose.  36.  446 
pentoses  in  urine,  443 
Tdpfer's    method    for    quantitative    analysis    of 

gastric  juice.  174 
Tfipfer's  reagent,  as  indicator,  133.  134,  164,  173 

preparation  of,  173 
Total  solids,  of  milk,  quantitative  determination 
of,  327 
of  urine,  quantitative  determination  of, 
483 
Total  sulphur  of  urine,  quantitative  determina- 
tion of,  348 
phosphorus    of    urine,    quantitative    deter- 
mination of,  334 
Trichloracetic  acid,  precipitation  of  protein  by, 

103,  120.  272 
Tricresol-peroxidase  reaction  (Kastle).  320 
Trimethyl-oxyethyl-ammonium    hydroxide      (see 

Choline,  212.  354,  337) 
Trioses.  19 
Tripeptides.  66,  93 
Triple  phosphate.  362.  408,  438,  476 
crystalline  form  of,  408 
formation  of.  408 
Trisaccharides,  19.  42 
Trommer's  test.  25 
Tropaeolin  O.  use  of,   as  indicator,    137 

preparation  of,  138 
Tropaeolin  OO,  use  of.  as  indicator,  133,  154.  135, 
157.  159 
preparation  of.  133.  138 
Tropaeolin  000,  use  of,  as  indicator,   137 

preparation  of,  138 
Trypsin  (see  also  Pancreatic  protease.  3,  12,  186) 
action  of,  upon  proteins,  66,  186,  189 
experiments  on,  190 
influence  of  alkalis  and  mineral  acids  upon, 

186 
in  stomach,  131,  169 

determination  of,  169 
nature  of,  186 
Trypsinogen.  6.  186 

activation  of,  6,  186,  196 
Tryptic  digestion,  66,  186 

influence  of  bile  on,  190 

most  favorable  reaction  for,  19  r 

temperature  for,  192 
products  of,  66,  186,  189 
Tryptic  proteolysis,  142,  i86 
Tryptophane,  63,  67,  68,  77.  98,  199 
bromine  water  test  for,  199 
formula  for,  77 

group  in  the  protein  molecule,  98 
Hopkins-Cole  reaction  for,  98 
mercury  compound  of,  preparation  of,  190 
occurrence  of,  as  a  decomposition  product  of 

protein,  65,  67.  68.  77 
occurrence   of.   as   an   end-product   of   pan» 
creatic  digestion,  186,  189 
Tuberculosis,  urochromogen  reaction  for,  433 
Tussah  silk  fibroin,  68 
Tyrosinase,  3 
Tyrosine,  3,  65,  67.  69.  74.  86,  97.  i86,  463 


6s6 


INDEX 


Tyrosine,  crystalline  form  of,  76 

experiments  on,  86 

formula  for,  77 

Hoffmann's  reaction  for,  86 

in  urinary  sediments,  463 

microscopical  examination  of,  86 

Morner's  test  for,  86 

occurrence  of,  6s,  67,  69,  I86 

Piria's  test  for,  86 

salts  of,  77 

separation  of,  from  leucine,  85 

solubility  of,  86 

sublimation  of,  86 
Tyrosine-sulphuric  acid,  86 

V.  Udrdnsky's  test  for  bile  acids,  208,  434 
Uffelmann's  reagent,  preparation  of,   170 

reaction  for  lactic  acid,  170 
Unknown  substances  in  urine,  454 
Unorganized  ferments,   i 

sediments  in  urine,  457,  458 
Unsaturated  acids,   177 
Uranium   acetate    method   for   determination   of 

total  phosphates  in  urine,  552 
Uracil,  125,  128,  133 

Wheeler-Johnson  reaction  for,   133 
Urate,  ammonium,  crystalline  form  of,  Plate  VI, 
opposite  p.  462 
sodium,  crystalline  form  of,  462 
Urates  in  urinary  sediments,  461 
Urea,  248,  274,  341,  372,  491 
crystalline  form  of,  372 
decomposition  of,   by  sodium   hypobromite, 

374.  496 
excretion  of,  372,  374 
experiments  on,  37s 
formation  of,  373 
formula  for,  372 
furfural  test  for,  377 
isolation  of,  from  the  urine,  375 
melting-point  of,  37s 

quantitative  determination  of,  in  blood,  274 
in  urine,  491-499 
Urea  nitrate,  374,  376 

crystalline  form  of,  374 
formula  for,  374 
oxalate,  374,  376 

crystalline  form  of,  376 
formula  for,  374 
Urease,  4 

decomposition  of  urea  by,  491 

preparation  of,  491 
quantitative  determination  of  urea  by,  274, 
491 
Uremia,  blood  in,  270,  271 
Urethral    filaments    in    urinary    sediments,    46s, 

473 
Uric  acid,  26,  127,  133,  137,  248,  270,  274,  341,  369, 
377-381,  460,  476.  510,  57 1.  573 
calculi,  476 

crystalline  form  of,  pure,  380 
endogenous,  378,  573 
exogenous,  378,  571 
experiments  on,  380 
formula  for,  377 
Ganassini's  test,  381 
in  blood,  248,  270,  274 
in  leukaemia,  379 


Uric  acid  in  urinary  sediments,  460 

crystalline  form  of,  Plate  V,  oppo- 
site p.  380,  461 
isolation  of,  from  the  urine,  380 
metabolism,  571,  573 
murexide  test  for,  380 
origin  of,  378 

quantitative  determination  of,  in  blood, 
Benedict's  method,  275 
Folin-Denis  method,  274 
in    urine,    microchemical    colo- 
rimetric  method,  510 
Folin-Shafler  method,  5  1 1 
Heintz  method,  s  12 
Kruger- Schmidt     method, 

513 
Uricometer  method,  5  13 
quantitative   determination   of,    Kruger 

and  Schmidt's  method  for,  s  13 
Hunter  and  Givens'  modification  of,  5  15 
reducing  power  of,  26,  379,  416,  417 
Ruhemann's  uricometer  method  for,  5  13 
Schiff's  reaction  for,  381 
Uricase,  5,   127 

experiments  on,   137 
Uricolytic  enzymes,  3,  5,  127 
experiments  on,  137 
Urinary  calculi,  475 

calcium  carbonate  in,  476 
oxalate  in,  476 
cholesterol  in,  478 
compound,  475 
cystine  in,  476 
fibrin  in,  478 
indigo  in,  478 
phosphates  in,  476 
scheme  for  chemical  analysis  of,  477 
simple,  475 

uric  acid  and  urates  in,  476 
urostealiths  in,  478 
xanthine  in,  476 
Urinary  concrements  (see  Urinary  calculi,  p.  475) 
Urinary  sediments,  457 

ammonium    magnesium    phosphate    in, 

458 
animal  parasites  in,  465,  474 
calcium  carbonate  in,  458,  459 
oxalate  in,  458 
phosphate  in,  458,  460 
sulphate  in,  458,  460 
casts  in,  465,  467 
cholesterol  in,  458,  463 
collection  of,  457 
cylindroids  in,  465,  472 
cystine  in,  458,  462 
epithelial  cells  in,  465 
erythrocytes  in,  465,  472 
fibrin  in,  465,  474 
foreign  substances  in,  465,  474 
hematoidin  and  bilirubin  in,  458,  464 
hippuric  acid  in,  458,  463 
indigo  in,  458,  464 
leucine  and  tyrosine  in,  458,  463 
magnesium  phosphate  in,  458,  464 
melanin  in,  458,  465 
micro-organisms  in,  465,  474 
organized,  457,  465 
pus  cells  in,  4<>5,  466 


INDEX 


637 


Urinary  sediments,  spermatozoa  in,  46s,  474 

tissue  debris  in,  465,  473 

unorganized,  4S7.  4S8 

urates  in,  458,  461 

urethral  filaments  in,  465,  473 

uric  acid  in,  458,  460 

xanthine  in,  458,  464 
Urination,  frequency  of,  36  i 
Urine,  359-563 

acetoacetic  acid  in,  412,  438,  539 

acetone  in,  412,  435,  538 

acidity  of,  361,  479,  577.  S8o,  582 

acid  fermentation  of,  363 

albumin  in,  412,  422,  S3i 

alkaline  fermentation  of,  362,  408 

allantoin  in,  369. '392,  5  18 

amino-acids  in,  369,  394,  502 

ammonia  in,  369,  402,  499 

aromatic  oxyacids  in,  369,  394 

benzoic  acid  in,  369,  39s 

bile  in,  412,  432 

blood  in,  412,  429 

calcium  in,  370,  409,  559 

carbonates  in,  370,  410 

chlorides  in,  370,  405 

collection  of,  367 

collection  and  preservation  of,  in  metabolism 

tests,  367,  565 
color  of,  359 

complete  analysis  of,  565 
conjugate  glycuronates  in,  412,  442 
creatine  in,  369,  384,  508,  509 
creatinine  in,  369,  381,  506,  574 
dextrose  in  (see  glucose,  412,  413,  S22) 
diacetic  acid  in  (see  acetoacetic  acid,  412,  438, 

539) 
electrical  conductivity  of,  366 
enzymes  in,  370,  397 
ethereal  sulphuric  acid  in,  369,  385,  547 
fat  in,  4  12,  444 
fluorides  in,  370,  411 
freezing-point  of,  365 
fructose  in,  412,  446 
galactose  in,  412,  446 
general  characteristics  of,  359 
globulin  in,  412,  426 
glucose  in,  412,  413,  522 
Haser's  coefficient  for  solids  in,  364,  483 
hematoporphyrin  in,  412,  444 
hippuric  acid  in,  369,  388,  5  19 
hydrogen  ion  concentration  of,  36  I,  480,  580 
as  influenced  by  diet,  580 
by  acid  and  alkali,  582 
hydrogea  peroxide  in,  370,  411 
|8-hydroxybutyric  acid  in,  412,  440,  539 
inorganic  physiological  constituents  of,  370, 

402 
inositol  in,  412,  450 
iron  in,  370,  410,  562 
lactose  in,  4  12,  444 
levulose  in  (see  fructose,  412,  446) 
laiose  in,  412,  451 
leucomaines  in,  370,  401 
Long's  coefficient  for  solids  in,  364,  483 
magnesium  in,  370,  409,  559 
melanin  in,  412,  451 

neutral  sulphur  compounds  in,  369,  393,  55  i, 
578 


Urine,  nitrates  in,  370,  411 

nucleoprotein  in,  369,  396,  412,  428 

odor  of,  36  I 

organic  physiological  constituents  of,  369 

oxalic  acid  in,  369,  391 

oxaluric  acid  in,  369.  397 

/9-oxybutyric    acid    in    (see     hydroxybutyric 
acid,  412,  440,  S39) 

pathological  constituents  of,  412 

paralactic  acid  in,  370,  398 

pentoses  in,  412,  443 

peptone  in,  412,  426 

phenaceturic  acid  in,  370,  398 

phosphates  in,  370,  406,  552 

phosphorized  compounds  in,  370,  399 

physiological  constituents  of,  369 

pigments  of,  359,  370,  399 

potassium  in,  370,  409,  s6i 

proteins  in,  412,  422 

proteoses  in,  412,  422,  426 

ptomaines  in,  370,  401 

purine  bases  in,  370,  401,  Si3 

quantitative  analysis  of,  479-5^3 

reaction  of,  361,  479,  S8o 

as  influenced  by  diet,  580 

by  acids  and  alkalies,  582 

silicates  in,  370,  411 

sodium  in,  370,  409,  56  I 

solids  of,  364,  483 

specific  gravity  of,  363 

sulphates  in,  370,  403»  S46 

transparency  of,  360 

unknown  substances  in,  412,  454 

urea  in,  369,  372,  491 

uric  acid  in,  369,  377,  5 10 

urinod  in,  361 

urorosein  in,  412,  452 

volatile  fatty  acids  in,  370,  397 

volume  of,  359 
Urinod,  361 
Urobilin,  359,  370,  399 

tests  for,  400 
Urobilinogen,  399 
Urocanic  acid,  398 
Urochrome,  359,  3"0,  399,  4^1 
Urochromogen,  412,  453 

reaction  (Weisz)  for  tuberculosis,  453 
Uroerythrin,  359,  370,  399.  421 
Uroferric  acid,  369,  392,  454 
Uroleucic  acid,  369,  395 
Urorosein,  412,  452 

reaction,  452 

tests  for,  452 

Valine,  65,  67,  69,  78 

Van  Slyke's  method  for  determination  of  total 
amino-acid    nitrogen, 
in  urine,  505 
in  blood,  377 
in  protein  hydrolysis, 
88 
Van  Slyke  and  CuUea's  method  for  urea  in  blood, 
274 
for  urea  in  urine,  49a 
Van  Slyke  and  McLeans  method  for  chlorides  in 

blood,  286 
Vegetable  amylase,  4,  11 
lipase,  5,   13 


638 


INDEX 


Vegetable  protease,  13 

sucrase,   14,   195,   199 
Vegetable  globulins,  93,  95,  108 
Vegetable  gums,  20 
Veith    lactometer,    determination  of    specific 

gravity  of  milk  by,  324 
Viscosity  test,  59 
Vitamines,  313 
Vitellin,  94,  95 

Volatile  fatty  acids,  212,  215,  370,  397 
Volhard-Arnold    method    for    determination    of 

chlorides,  556 
Volhard-Harvey    method    for    determination    of 

chlorides,  557 
Volume  of  the  urine,  359 

Water  at  meals,  influence  of,  138,   i8s,  402,  574 

softened,  57 
Water  test  meal,   161 
Water,  influence  of  on  metabolism,  574 
Wax  myrtle,   181 

Waxy  casts  in  urinary  sediments,  465,  470 
Weber's  guaiac  test  for  blood  in  feces,  235 
Weinland,  formation  of  fat  from  protein,   179 
Weisz's  urochromogen  reaction  for  tuberculosis, 

453 
Welker's  electrical  bath,  494 

modified  method  for  purine  bases,  s  15 
Welker  and  Marsh  method  for  deproteinizing  milk, 

329 
Welker    and    Tracy    method    for    deproteinizing 

urine,  485 
Weyl's  test  for  creatinine,  385 
Wheeler- Johnson  reaction  for  uracil  and  cytosine, 

133 
White  fibrous  connective  tissue,  331 

experiments  on,  332 
Wiechowski-Handovsky     method     for     determi- 
nation of  allantoin  in  urine,  518 
Wilkinson  and  Peters'  test,  321 
Wirsing's  test  for  urobilin,  401 
Witchs'  milk,  317 

Wohlgemuths'    method    for    quantitative    deter- 
mination of  amylolytic  activity,   192 
Author's  modification  of,  239 


Wolter's    method   for   determination    of   iron    in 
urine,  562 

Xanthine,  126,  127,  132,  346,  354,  401 

bases  (see  Purine  bases,  pp.  126,  346,  401) 

crystalline  form  of,  344 

formula  for,  127,  346 

in  urinary  sediments,  458,  464 

isolation  of,  from  meat  extract,  35 1 

silver  nitrate,  351 

crystalline  form  of,  351 
test,  351 
.    tests  for,   132 

Weidel's  reaction  for,  132 
Xanthophylls,  319 
Xanthoproteic  reaction,  98 
Xanthinoxidase,  s 
Xylose,  20,  38 

orcinol  reaction  on,  38 

phenylhydrazine  reaction  on,  38 

Tollens'  reaction  on,  38 

Yeast,  enzymes  of,  2 

fermentation  by,  42 
nucleoprotein  of,  128 
preparation  of,   128 
tests  on,  129 
nucleic  acid  of,   125,  130 
formula  for,  125 
preparation  of,   130 
tests  on,   130 
Yellow  elastic  connective  tissue,  33;^ 
composition  of,  334 
experiments  on,  334 

Zappert  slide,  307 
Zein,  67,  69,  93,  95,  III 

decomposition  of,  67,  69 
Zeller's  test  for  melanin,  452 
V.  Zeynek  and  Nencki's  hemin  test,  266,  430 
Zikel  pektoscope,  365 
Zymase,  classification  of,  4 

preparation  of,  2 
Zymo-exciter,  7 
Zymogen,  6,  186 


